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PMID:7191 | Microbial formation and degradation of dimethylamine. | Dimethylamine was formed from trimethylamine in soils of different pH values. The rate of disappearance of the secondary amine from soil was affected by pH and was markedly reduced under anaerobiosis. The accumulation of dimethylamine in cultures of Micrococcus sp. provided with trimethylamine depended on the nitrogen sources available to the bacterium but was not greatly influenced by the C-N ratio of the medium. Dimethylamine and nitrite accumulated in large amounts at pH 6.0 to 8.0 in cultures containing the tertiary amine and nitrate, but dimethylnitrosamine was apparently not produced. | Microbial formation and degradation of dimethylamine. Dimethylamine was formed from trimethylamine in soils of different pH values. The rate of disappearance of the secondary amine from soil was affected by pH and was markedly reduced under anaerobiosis. The accumulation of dimethylamine in cultures of Micrococcus sp. provided with trimethylamine depended on the nitrogen sources available to the bacterium but was not greatly influenced by the C-N ratio of the medium. Dimethylamine and nitrite accumulated in large amounts at pH 6.0 to 8.0 in cultures containing the tertiary amine and nitrate, but dimethylnitrosamine was apparently not produced. | [
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PMID:7192 | Toxicity of ammonia to algae in sewage oxidation ponds. | Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond. | Toxicity of ammonia to algae in sewage oxidation ponds. Ammonia, at concentrations over 2.0 mM and at pH values over 8.0, inhibits photosynthesis and growth of Scenedesmus obliquus, a dominant species in high-rate sewage oxidation ponds. Photosynthesis of Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum is also susceptible to ammonia inhibition. Dark respiration and cell morphology were unaffected by any combination of pH and ammonia concentrations tested, thus limiting the apparent effect to inhibition of the normal function of the chloroplasts. Methylamine had the same effect as ammonia, and its penetration into the cells was found to be pH dependent. Therefore, the dependence of toxicity of amines to algae on pH apparently results from the inability to penetrate the cell membrane in the ionized form. When operated at 120-h detention time of raw wastewater, the high-rate oxidation pond maintained a steady state with respect to algal growth and oxygen concentration, and the concentration of ammonia did not exceed 1.0 mM. Shifting the pond to 48-h detention time caused an increase in ammonia concentration in the pond water to 2.5 mM, and the pond gradually turned anaerobic. Photosynthesis, which usually elevates the pH of the pond water to 9.0 to 10.0, could not proceed beyond pH 7.9 because of the high concentration of ammonia, and the algal population was washed out and reduced to a concentration that could maintain a doubling time of 48 h without photosynthesis bringing the pH to inhibitory levels. Under these conditions, the pH of the bond becomes a factor that limits the operational efficiency of the oxidation pond. | [
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PMID:7193 | Production of extracellular alpha-glucosidase by a thermophilic Bacillus species. | Production of extracellular alpha-glucosidase was studied with strain KP 1006 of a new species of thermophilic Bacillus, which was isolated from soil samples by enrichment at 65 C. alpha-Glucosidase production was maximum at 60 C and at an initial pH of 6.5. The final enzyme yield was increased by starch, maltose, glycerol, peptone, and yeast extract but reduced by acetate and gluconate, alpha-Glucosidase was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. At a midpoint of this period, the enzyme appeared in the culture broth, and its level increased until the end of the stationary phase. | Production of extracellular alpha-glucosidase by a thermophilic Bacillus species. Production of extracellular alpha-glucosidase was studied with strain KP 1006 of a new species of thermophilic Bacillus, which was isolated from soil samples by enrichment at 65 C. alpha-Glucosidase production was maximum at 60 C and at an initial pH of 6.5. The final enzyme yield was increased by starch, maltose, glycerol, peptone, and yeast extract but reduced by acetate and gluconate, alpha-Glucosidase was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. At a midpoint of this period, the enzyme appeared in the culture broth, and its level increased until the end of the stationary phase. | [
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PMID:7194 | Factors influencing the production of cellulases by Sporotrichum thermophile. | Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi. | Factors influencing the production of cellulases by Sporotrichum thermophile. Cellulase production and growth of a strain of Sporotrichum thermophile were studied by using a mineral salts medium supplemented with yeast extract and insoluble cellulose. The effects of cultural conditions, such as pH, nitrogen source, substrate concentration, and temperature, were examined. Maximum production of C1 and CX cellulases occurred at 45 C in 2 to 4 days, in the presence of 1% Solka/Floc as substrate, when NaNO3 or urea used as sources of nitrogen. Under these conditions, cellulolytic activity of culture filtrates appeared to be similar to that reported for Trichoderma viride grown in a favorable environment. However, comparable yields of cellulase were produced by S. thermophile in less than one-quarter the time required by mesophilic fungi. | [
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PMID:7195 | Degradation of [8,9,-14C]endosulfan by soil microorganisms. | Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur. | Degradation of [8,9,-14C]endosulfan by soil microorganisms. Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur. | [
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PMID:7196 | Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultrahigh temperature. | The heat-stable extracellular protease of Pseudomonas sp. (isolate MC60) was investigated. Heat resistance of the enzyme in milk at sterilization temperature was dependent on the presence of Ca2+. The half-life of the enzyme at ultrahigh temperature (149 C) in skim milk or milk-salts buffer with Ca2+ was approximately 7.0 s. Treatment of milk with chelators completely removed the heatstabilizing effect of milk. The enzyme was partially purified by ammonium sulfate precipitation and column chromatography on Sephadex G-100. At 21 C the enzyme retained greater than 85% activity after exposure to pH values between 5 and 10. Enzyme activity was reduced by metal chelating agents. Both Ca2+ and Zn2+ were required for optimal enzyme activity. Molecular weight was estimated at 48,000 by gel filtration. | Stabilization of a psychrotrophic Pseudomonas protease by calcium against thermal inactivation in milk at ultrahigh temperature. The heat-stable extracellular protease of Pseudomonas sp. (isolate MC60) was investigated. Heat resistance of the enzyme in milk at sterilization temperature was dependent on the presence of Ca2+. The half-life of the enzyme at ultrahigh temperature (149 C) in skim milk or milk-salts buffer with Ca2+ was approximately 7.0 s. Treatment of milk with chelators completely removed the heatstabilizing effect of milk. The enzyme was partially purified by ammonium sulfate precipitation and column chromatography on Sephadex G-100. At 21 C the enzyme retained greater than 85% activity after exposure to pH values between 5 and 10. Enzyme activity was reduced by metal chelating agents. Both Ca2+ and Zn2+ were required for optimal enzyme activity. Molecular weight was estimated at 48,000 by gel filtration. | [
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PMID:7197 | N-Nitrosamine formation by cultures of several microorganisms. | Of 38 pure cultures of microorganisms tested, only one, Pseudomonas stutzeri, was capable of forming dimethylnitrosamine from dimethylamine and nitrite during growth. Resting cells of P. stutzeri, Cryptococcus terreus, Escherichia coli, and Xanthomonas campestris formed dimethylnitrosamine, although no nitrosamine was found in growing cultures of the latter three organisms. No nitrosamine was produced by either growing cultures or resting-cell suspensions of Pseudomonas fragi or Proteus mirabilis. Boiled cells of P. stutzeri, but not those of C. terreus, E. coli, and X. campestris, formed dimethylnitrosamine, and this nitrosamine was also produced by extracts of E. coli cells at pH 5.0. | N-Nitrosamine formation by cultures of several microorganisms. Of 38 pure cultures of microorganisms tested, only one, Pseudomonas stutzeri, was capable of forming dimethylnitrosamine from dimethylamine and nitrite during growth. Resting cells of P. stutzeri, Cryptococcus terreus, Escherichia coli, and Xanthomonas campestris formed dimethylnitrosamine, although no nitrosamine was found in growing cultures of the latter three organisms. No nitrosamine was produced by either growing cultures or resting-cell suspensions of Pseudomonas fragi or Proteus mirabilis. Boiled cells of P. stutzeri, but not those of C. terreus, E. coli, and X. campestris, formed dimethylnitrosamine, and this nitrosamine was also produced by extracts of E. coli cells at pH 5.0. | [
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PMID:7198 | Influence of sewage discharge on nitrogen fixation and nitrogen flux from coral reefs in Kaneohe Bay, Hawaii. | Nitrogen fixation was investigated in Kaneohe Bay, Oahu, Hawaii, a subtropical eutrophic estuary, by using the acetylene reduction technique on algal samples. No active, planktonic, N2-fixing blue-green algae or bacteria were observed. However, Calothrix and Nostoc capable of fixing N2 were cultured from navigational buoys and dead coral heads. Nitrogen fixation associated with these structures was greater in the middle sector than in the south and north sectors of the estuary. Experiments demonstrated that the fixation was photosynthetically dependent. Examination of the data showed that there was no significant correlation between rates of nitrogen fixation and concentration of combined nitrogen compounds in the Bay water. Fixation was significantly correlated to the inorganic N/P (atomic) ratio in the south and middle sectors but not in the north sector. The nutrient data indicate there was a flux of combined nitrogen, but not phosphate, from the reef flats. | Influence of sewage discharge on nitrogen fixation and nitrogen flux from coral reefs in Kaneohe Bay, Hawaii. Nitrogen fixation was investigated in Kaneohe Bay, Oahu, Hawaii, a subtropical eutrophic estuary, by using the acetylene reduction technique on algal samples. No active, planktonic, N2-fixing blue-green algae or bacteria were observed. However, Calothrix and Nostoc capable of fixing N2 were cultured from navigational buoys and dead coral heads. Nitrogen fixation associated with these structures was greater in the middle sector than in the south and north sectors of the estuary. Experiments demonstrated that the fixation was photosynthetically dependent. Examination of the data showed that there was no significant correlation between rates of nitrogen fixation and concentration of combined nitrogen compounds in the Bay water. Fixation was significantly correlated to the inorganic N/P (atomic) ratio in the south and middle sectors but not in the north sector. The nutrient data indicate there was a flux of combined nitrogen, but not phosphate, from the reef flats. | [
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PMID:7199 | Cultivation of mycoplasmas in a modified tissue culture medium. | A new medium, which contained a chemically defined tissue culture base ("medium 199"), was developed for the cultivation of mycoplasmas. When supplemented with albumin, glucose, serum, and yeast extract, the new medium adequately supported the growth of Mycoplasma and Acholeplasma species. | Cultivation of mycoplasmas in a modified tissue culture medium. A new medium, which contained a chemically defined tissue culture base ("medium 199"), was developed for the cultivation of mycoplasmas. When supplemented with albumin, glucose, serum, and yeast extract, the new medium adequately supported the growth of Mycoplasma and Acholeplasma species. | [
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PMID:7215 | Further evidence for the transmission of Mansonella ozzardi by Simulium amazonicum in Brazil. | The transmission of Mansonella ozzardi was studied in two rubber collecting villages on the River Purus, state of Amazonas, Brazil. Haematophagous insects were collected from human and bovine baits during the day and night: 687 Mansonia amazonensis and 154 Culicoides sp. indet. were free from infection with M. ozzardi. The former species is probably not a vector, but the low numbers of culicoides dissected preclude any determination of its vector status. Thirty-five (0-99%) of 3530 Simulium amazonicum dissected were found naturally infected with larvae of M. ozzardi. Two hundred and ninety-nine M. amazonensis and 280 S. amazonicum were experimentally infected with M. ozzardi by feeding on volunteers. Microfilariae were detected in the blood meals of both species but no developing larvae were found in the thoracic muscles of M. amazonensis, confirming its non-vector status; 7-1% of the S. amazonicum dissected had filarial larvae in the thorax. There was a statistically significant difference between this rate and the natural rate of infection in this region, verifying that penetration of the thoraric muscles by M. ozzardi had occurred in the experimental infection. The data confirm the observation of Cerqueira (1959) that S. amazonicum transmits M. ozzardi in Brazil. | Further evidence for the transmission of Mansonella ozzardi by Simulium amazonicum in Brazil. The transmission of Mansonella ozzardi was studied in two rubber collecting villages on the River Purus, state of Amazonas, Brazil. Haematophagous insects were collected from human and bovine baits during the day and night: 687 Mansonia amazonensis and 154 Culicoides sp. indet. were free from infection with M. ozzardi. The former species is probably not a vector, but the low numbers of culicoides dissected preclude any determination of its vector status. Thirty-five (0-99%) of 3530 Simulium amazonicum dissected were found naturally infected with larvae of M. ozzardi. Two hundred and ninety-nine M. amazonensis and 280 S. amazonicum were experimentally infected with M. ozzardi by feeding on volunteers. Microfilariae were detected in the blood meals of both species but no developing larvae were found in the thoracic muscles of M. amazonensis, confirming its non-vector status; 7-1% of the S. amazonicum dissected had filarial larvae in the thorax. There was a statistically significant difference between this rate and the natural rate of infection in this region, verifying that penetration of the thoraric muscles by M. ozzardi had occurred in the experimental infection. The data confirm the observation of Cerqueira (1959) that S. amazonicum transmits M. ozzardi in Brazil. | [
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PMID:7216 | [Studies on the properties of acid erythrocyte phosphatase in sheep and the isoenzymes of sheep and goat acid erythrocyte phosphatase]. | Ovine erythrocytic acid phosphatase showed two peaks of activity at pH 5.0 and 5.7 in acetate buffer with p-nitrophenylphosphate as substrate. The enzyme was only slightly inhibited by fluoride and L-phenylalanine, but high concentrations of urea strongly inhibited it. Activity of the enzyme was greater in goat erythrocytes than in sheep. By means of starch electrophoresis, three isoenzymes belonging to nine types were separated from the ovine enzymes, while three isoenzymes of five types were present in goats. Electrophoresis in polyacrylamide gel was suitable for detecting the rapidly migrating isoenzymes. | [Studies on the properties of acid erythrocyte phosphatase in sheep and the isoenzymes of sheep and goat acid erythrocyte phosphatase]. Ovine erythrocytic acid phosphatase showed two peaks of activity at pH 5.0 and 5.7 in acetate buffer with p-nitrophenylphosphate as substrate. The enzyme was only slightly inhibited by fluoride and L-phenylalanine, but high concentrations of urea strongly inhibited it. Activity of the enzyme was greater in goat erythrocytes than in sheep. By means of starch electrophoresis, three isoenzymes belonging to nine types were separated from the ovine enzymes, while three isoenzymes of five types were present in goats. Electrophoresis in polyacrylamide gel was suitable for detecting the rapidly migrating isoenzymes. | [
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PMID:7218 | [Origination and importance of glycolysis for malignomas and utilization of this property in the chemotherapy of cancer (author's transl)]. | Glycolysis is not of importance for the process of carcinogenesis. It is very likely, however, that certain molecular-biological and genetic changes are produced which enable the malignant cell to develop an intensive glycolysis, for instance, to form specialized glycolytic isoenzymes already during oncogenesis, and may possible become effective in the primary tumour. As soon as the capacity of the cancer cell to intensive aerobic and anaerobic glycolysis has become manifest, this process is an irreversible one. The extent of glycolysis of a malignoma is greatly dependent on the degree of its dedifferentiation and vascularization (glucose supply), although a direct correlation between growth and the amount of lactic acid formed does not seem to exist. However, a certain utilization of glucose is essential for cell proliferation (supply of basic substances). In many cases there is a correlation between the extent of glycolysis measurable under optimal conditions in vitro (glycolytic power) in a malignant tumour and its growth rate recognizable in vivo. The formation of a strong capacity for glucose degradation via the Embden-Meyerhof pathway that cannot be fully utilized by the whole tumour in vivo is first of all designed to ensure survival and proliferation of cells even at extremely low levels of glucose supply. This process can be regarded as an adaptation of cancer cells to a situation of unsufficient supply. This circumstance endows the cancer cell with an essential advantage over the normal cell which enables or even promotes its invasive and destructive growth and metastatic dissemination. In this respect they differ, for instance, from benignant neoplasms. The possibility is discussed to control neoplastic growth by adjusting an optimal pH difference between normal and tumour tissue by combined administration of detoxicated drugs which are converted to their toxic forms only in the tumour by means of strongly pH-dependent exogenous enzymes. | [Origination and importance of glycolysis for malignomas and utilization of this property in the chemotherapy of cancer (author's transl)]. Glycolysis is not of importance for the process of carcinogenesis. It is very likely, however, that certain molecular-biological and genetic changes are produced which enable the malignant cell to develop an intensive glycolysis, for instance, to form specialized glycolytic isoenzymes already during oncogenesis, and may possible become effective in the primary tumour. As soon as the capacity of the cancer cell to intensive aerobic and anaerobic glycolysis has become manifest, this process is an irreversible one. The extent of glycolysis of a malignoma is greatly dependent on the degree of its dedifferentiation and vascularization (glucose supply), although a direct correlation between growth and the amount of lactic acid formed does not seem to exist. However, a certain utilization of glucose is essential for cell proliferation (supply of basic substances). In many cases there is a correlation between the extent of glycolysis measurable under optimal conditions in vitro (glycolytic power) in a malignant tumour and its growth rate recognizable in vivo. The formation of a strong capacity for glucose degradation via the Embden-Meyerhof pathway that cannot be fully utilized by the whole tumour in vivo is first of all designed to ensure survival and proliferation of cells even at extremely low levels of glucose supply. This process can be regarded as an adaptation of cancer cells to a situation of unsufficient supply. This circumstance endows the cancer cell with an essential advantage over the normal cell which enables or even promotes its invasive and destructive growth and metastatic dissemination. In this respect they differ, for instance, from benignant neoplasms. The possibility is discussed to control neoplastic growth by adjusting an optimal pH difference between normal and tumour tissue by combined administration of detoxicated drugs which are converted to their toxic forms only in the tumour by means of strongly pH-dependent exogenous enzymes. | [
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PMID:7219 | [The influence of role induction on the success of systematic desensitization of socially anxious students (author's transl)]. | The so-called nonspecific factors involved in therapy have received little attention up to the present - namely, that of role induction (preparing the clients for treatment by informing them about the rationale of treatment, the treatment process, and their part in therapy). For this reason, we constructed an induction text for systematic desensitization according to the principles of instructional psychology (Ausubel), and proceeded to test it for understandability and therapeutic effectiveness. With control group design which assured the realization of the independent variable of role induction, the effective results of desensitization with role induction were compared both with those of simple desensitization and those of a waiting control group. The resultant differences in the group with role induction indicate the additional advantages of this technique for therapy. Essentially, role induction seems to manifest itself in terms of a contribution to more active, independent role formation on the part of the client. | [The influence of role induction on the success of systematic desensitization of socially anxious students (author's transl)]. The so-called nonspecific factors involved in therapy have received little attention up to the present - namely, that of role induction (preparing the clients for treatment by informing them about the rationale of treatment, the treatment process, and their part in therapy). For this reason, we constructed an induction text for systematic desensitization according to the principles of instructional psychology (Ausubel), and proceeded to test it for understandability and therapeutic effectiveness. With control group design which assured the realization of the independent variable of role induction, the effective results of desensitization with role induction were compared both with those of simple desensitization and those of a waiting control group. The resultant differences in the group with role induction indicate the additional advantages of this technique for therapy. Essentially, role induction seems to manifest itself in terms of a contribution to more active, independent role formation on the part of the client. | [
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PMID:7220 | Cell populations in a renal lesion produced by local injection of xenogeneic spleen cells in cyclophosphamide-treated rats. | The frequency and distribution of donor and host lymphoid cells in different stages of a lesion produced by injecting mouse spleen cells beneath the renal capsules of rats treated 24 h previously with cyclophosphamide have been studied by immunofluorescent staining with species-specific anti-lymphocyte sera. Donor cells were predominant in the early stages of the reaction and penetrated the outer part of the renal cortex, but by day 7 when the lesion reached its maximum extent most of the infiltrating cells were of host origin. Donor cells never extended deeply into the kidney and they were not uniformly intermingled with the main mass of host cells but their presence seemed to be necessary for the maintenance of the reaction, since it began to decline when donor cells were no longer detectable in the injected kidney. | Cell populations in a renal lesion produced by local injection of xenogeneic spleen cells in cyclophosphamide-treated rats. The frequency and distribution of donor and host lymphoid cells in different stages of a lesion produced by injecting mouse spleen cells beneath the renal capsules of rats treated 24 h previously with cyclophosphamide have been studied by immunofluorescent staining with species-specific anti-lymphocyte sera. Donor cells were predominant in the early stages of the reaction and penetrated the outer part of the renal cortex, but by day 7 when the lesion reached its maximum extent most of the infiltrating cells were of host origin. Donor cells never extended deeply into the kidney and they were not uniformly intermingled with the main mass of host cells but their presence seemed to be necessary for the maintenance of the reaction, since it began to decline when donor cells were no longer detectable in the injected kidney. | [
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PMID:7221 | Facilitation of the growth of an allogeneic tumour by suppressor cells in newborn rats. | The intravenous injection of as few as 15 Walker tumour cells into newborn rats consistently resulted in the development of pulmonary metastases and the death of the recipient within 2 weeks. Neither the outcome of tumour cell injection nor the interval until death could be modified by transferring 2 x 10(7) lymphocytes from tumour-immune adult rats to the neonataal hosts. In contrast with this failure to transfer adoptive anti-tumour immune responses to intact recipients, the administration of 350 rad irradiation before transfer of 10(6) immune lymphocytes constantly afforded protection against inoculated tumour cells. The simultaneous transfer of neonatal thymus cells with immune lymphocytes interfered with the establishment of an adoptive response in the irradiated newborn. Intiation of a graft-versus-host response in F1 hybrid neonates by injecting parental strain lymphocytes conferred resistance to tumour growth of the recpient, the magnitude of this effect increasing with the strength of the graft-versus-host reaction. | Facilitation of the growth of an allogeneic tumour by suppressor cells in newborn rats. The intravenous injection of as few as 15 Walker tumour cells into newborn rats consistently resulted in the development of pulmonary metastases and the death of the recipient within 2 weeks. Neither the outcome of tumour cell injection nor the interval until death could be modified by transferring 2 x 10(7) lymphocytes from tumour-immune adult rats to the neonataal hosts. In contrast with this failure to transfer adoptive anti-tumour immune responses to intact recipients, the administration of 350 rad irradiation before transfer of 10(6) immune lymphocytes constantly afforded protection against inoculated tumour cells. The simultaneous transfer of neonatal thymus cells with immune lymphocytes interfered with the establishment of an adoptive response in the irradiated newborn. Intiation of a graft-versus-host response in F1 hybrid neonates by injecting parental strain lymphocytes conferred resistance to tumour growth of the recpient, the magnitude of this effect increasing with the strength of the graft-versus-host reaction. | [
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PMID:7222 | Suppressor cells in homograft tolerant rats. | If sufficient normal syngeneic lymphocytes to effect skin graft rejection are transferred to homograft tolerant rats, a prolonged period elapses before lymphoid cells from the recipient acquire normal levels of GvH responsiveness against tissues of which the donor was previously tolerant (Silvers and Billingham, 1970; Elkins, 1972; Miyamoto and McCullagh, 1974). Although the ability of lymphoid populations of such animals to mount GvH reactions can be demonstrated to reside in donor type cells during the weeks immediately after transfer, reactive cells are ultimately derived from the host itself (Elkins, 1973; Miyamoto and McCullagh, 1974). Not only are lymphoid cells from tolerant rats which have been injected recently with normal lymphocytes poorly responsive in a GvH assay, but they have been observed in some experiments to suppress the GvH activity of normal syngeneic lymphoid cells (Elkins, 1972; Atkins and Ford, 1972). It is not clear whether the cells mediating suppression of the normal lymphocytes were derived from the tolerant host itself or, alternatively, from the normal lymphocytes injected into it to terminate the tolerant state. The present experiments sought to delineate the origin of any suppressor cells within populations of lymphocytes collected from rats in which tolerance had recently been terminated. The indicate that suppression of the normal donor cells within such populations may be exerted by cells derived from the tolerant host. | Suppressor cells in homograft tolerant rats. If sufficient normal syngeneic lymphocytes to effect skin graft rejection are transferred to homograft tolerant rats, a prolonged period elapses before lymphoid cells from the recipient acquire normal levels of GvH responsiveness against tissues of which the donor was previously tolerant (Silvers and Billingham, 1970; Elkins, 1972; Miyamoto and McCullagh, 1974). Although the ability of lymphoid populations of such animals to mount GvH reactions can be demonstrated to reside in donor type cells during the weeks immediately after transfer, reactive cells are ultimately derived from the host itself (Elkins, 1973; Miyamoto and McCullagh, 1974). Not only are lymphoid cells from tolerant rats which have been injected recently with normal lymphocytes poorly responsive in a GvH assay, but they have been observed in some experiments to suppress the GvH activity of normal syngeneic lymphoid cells (Elkins, 1972; Atkins and Ford, 1972). It is not clear whether the cells mediating suppression of the normal lymphocytes were derived from the tolerant host itself or, alternatively, from the normal lymphocytes injected into it to terminate the tolerant state. The present experiments sought to delineate the origin of any suppressor cells within populations of lymphocytes collected from rats in which tolerance had recently been terminated. The indicate that suppression of the normal donor cells within such populations may be exerted by cells derived from the tolerant host. | [
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PMID:7223 | Efficacy of two minor tranquilizers in relieveing symptoms of functional gastrointestinal distress. | Two minor tranquilizers, diazepam and lorazepam were significantly better than a placebo preparation in relieving symptoms of functional gastrointestinal distress in 28 patients who took part in a double-blind cross-over trial. The beneficial therapeutic effect may be group- rather than preparation-specific. Both agents were also of significant benefit to the sub-group of patients with aerophagy as a major symptom. | Efficacy of two minor tranquilizers in relieveing symptoms of functional gastrointestinal distress. Two minor tranquilizers, diazepam and lorazepam were significantly better than a placebo preparation in relieving symptoms of functional gastrointestinal distress in 28 patients who took part in a double-blind cross-over trial. The beneficial therapeutic effect may be group- rather than preparation-specific. Both agents were also of significant benefit to the sub-group of patients with aerophagy as a major symptom. | [
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PMID:7224 | Hydrallazine and beta-blockade in refractory hypertension with characterization of acetylator phenotype. | In 30 refractory hypertensives a hydrallazine beta blocker combination was added to or substituted for previous antihypertensives. Over a mean period of 12 months a good or satisfactory blood pressure response resulted in 12 patients each, while six others had an unsatisfactory outcome. (Good = diastolic pressure (DP) less than 95 mmHg; Satisfactory = deltaDP greater than 15 mmHg or DP 95-105 mmHg; Unsatisfactory = DP greater than 105 mmHg or deltaDP less than 15mmHg.) Twelve of the patients had significant renal disease with serum creatinine greater than 2 mg/100 ml, but in these there was no evidence that renal hydrallazine retention potentiated an antihypertensive effect. Those with an unsatisfactory response were receiving slightly higher doses hydrallazine and propranol when compared with good responders. The average dose of hydrallazine was 258 mg/day and of propranolol 308 mg/day. Transient headache was not uncommon at the commencement of hydrallazine therapy. Angina and vertebro-basilar insufficiency were each aggravated in one patient, but resolved with dosage adjustment. A lupuslike rash developed in one patient, a slow acetylator on 300 mg hydrallazine/day who had received a total of 92 g over eleven months. The genetically determined acetylator phenotype was assessed in 75 subjects. A little over one third were found to be rapid acetylators. Those with slow acetylator phenotype did not show a more favourable phenotype did not show a more favourable blood-pressure response to equivalent doses of hydrallazine. | Hydrallazine and beta-blockade in refractory hypertension with characterization of acetylator phenotype. In 30 refractory hypertensives a hydrallazine beta blocker combination was added to or substituted for previous antihypertensives. Over a mean period of 12 months a good or satisfactory blood pressure response resulted in 12 patients each, while six others had an unsatisfactory outcome. (Good = diastolic pressure (DP) less than 95 mmHg; Satisfactory = deltaDP greater than 15 mmHg or DP 95-105 mmHg; Unsatisfactory = DP greater than 105 mmHg or deltaDP less than 15mmHg.) Twelve of the patients had significant renal disease with serum creatinine greater than 2 mg/100 ml, but in these there was no evidence that renal hydrallazine retention potentiated an antihypertensive effect. Those with an unsatisfactory response were receiving slightly higher doses hydrallazine and propranol when compared with good responders. The average dose of hydrallazine was 258 mg/day and of propranolol 308 mg/day. Transient headache was not uncommon at the commencement of hydrallazine therapy. Angina and vertebro-basilar insufficiency were each aggravated in one patient, but resolved with dosage adjustment. A lupuslike rash developed in one patient, a slow acetylator on 300 mg hydrallazine/day who had received a total of 92 g over eleven months. The genetically determined acetylator phenotype was assessed in 75 subjects. A little over one third were found to be rapid acetylators. Those with slow acetylator phenotype did not show a more favourable phenotype did not show a more favourable blood-pressure response to equivalent doses of hydrallazine. | [
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PMID:7225 | Neurotic and psychotic states attributed to Thai "Phii Pob" spirit possession. | In Thailand, the term "Phii" refers to spirits and ghosts to which are generally attributed power over human beings. In the present, spirit possession is usually found in rural areas. However among several spirits, there is one particularly interesting spirit selected for this presentation, the phii pob. The "Phii Pob" is a common spirit in the Northeast, North and some provinces in the Central part of Thailand (Suwanlert, 1972). Thai believers consider that the phii pob differs from other spirits. It originates in a living person, then conceals itself in the body of that person, who is called th originating host; it is believed that the phii pob hiding within a person can leave the body of that person to possess another, who is called the possessed host. The victims almost always are females. | Neurotic and psychotic states attributed to Thai "Phii Pob" spirit possession. In Thailand, the term "Phii" refers to spirits and ghosts to which are generally attributed power over human beings. In the present, spirit possession is usually found in rural areas. However among several spirits, there is one particularly interesting spirit selected for this presentation, the phii pob. The "Phii Pob" is a common spirit in the Northeast, North and some provinces in the Central part of Thailand (Suwanlert, 1972). Thai believers consider that the phii pob differs from other spirits. It originates in a living person, then conceals itself in the body of that person, who is called th originating host; it is believed that the phii pob hiding within a person can leave the body of that person to possess another, who is called the possessed host. The victims almost always are females. | [
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PMID:7231 | The effects of acidosis and alkalosis on coronary flow and cardiac nucleotide metabolism. | The changes of the coronary flows and of the cardiac nucleotide metabolism during acidosis and during alkalosis were studied in 50 perfused guinea pig hearts with and without hypoxia. At pH 7.0 the coronary flows increased, and at pH 7.8 a significant reduction of the flows took place. At 20% O2, acidosis elicited a further flow increase, whereas alkalosis inhibited the flow increase produced by hyoxia. The increases after adenosine injections and after coronary occlusions were greater during acidosis and smaller during alkalosis than at pH 7.4. The cardiac nucleotide contents did not clearly differ from the controls whereas adenosine exhibits higher levels in acidotic hearts. Alkalosis always induced a decreased production of adenine nucleoside irrespective of the presence or the absence of hypoxia. At 20% O2 a decreased ATP level and increased ADP- and CrP-contents could be observed during alkalosis. | The effects of acidosis and alkalosis on coronary flow and cardiac nucleotide metabolism. The changes of the coronary flows and of the cardiac nucleotide metabolism during acidosis and during alkalosis were studied in 50 perfused guinea pig hearts with and without hypoxia. At pH 7.0 the coronary flows increased, and at pH 7.8 a significant reduction of the flows took place. At 20% O2, acidosis elicited a further flow increase, whereas alkalosis inhibited the flow increase produced by hyoxia. The increases after adenosine injections and after coronary occlusions were greater during acidosis and smaller during alkalosis than at pH 7.4. The cardiac nucleotide contents did not clearly differ from the controls whereas adenosine exhibits higher levels in acidotic hearts. Alkalosis always induced a decreased production of adenine nucleoside irrespective of the presence or the absence of hypoxia. At 20% O2 a decreased ATP level and increased ADP- and CrP-contents could be observed during alkalosis. | [
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PMID:7235 | Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase. | A simple radioactive-substrate assay for prostaglandin synthase (EC 1.14.99.1), which uses t.l.c. to measure simultaneously different prostaglandins synthesized from one precursor substrate, was developed. Rabbit kidney-medulla prostaglandin synthase catalyses the formation of prostaglandin E2, prostaglandin F2alpha and prostaglandin D2 from arachidonic acid. Fractionation of crude homogenates indicated that the microsomal fraction possessed the highest specific activity of prostaglandin synthase, whereas the soluble fraction exhibited little enzyme activity but rather contained a heat-labile inhibitory macromolecular factor(s), which might be attributed to the serum albumin present in this fraction. The microsomal fraction possessed low intrinsic enzyme activity, but the actvity could be fully stimulated by the presence of both GSH (reduced glutathione) and a phenolic cofactor. Only cysteine could partially replace GSH, whereas other thiols were inactive and some were even inhibitory. A variety of phenolic compounds, including catecholamines, dopamine (3,4-dihydroxyphenethylamine), 5-hydroxytryptamine and quinol, were active in stimulating prostaglandin synthase. In all cases, the stimulation was reflected in the synthesis of all three prostaglandins with ratios not significantly altered by different phenolic cofactors. The synthesis of each of the different prostaglandins appeared to have similar pH optima. The enzyme system was not inhibited by thiol-group inhibitors or a variety of metal chelators except for cyanide and 8-hydroxyquinoline. Characterization of the kidney-medulla prostaglandin synthase system indicated that it exhibited properties similar to those of the enzyme system present in seminal vesicles. | Biosynthesis of prostaglandins in rabbit kidney medulla. Properties of prostaglandin synthase. A simple radioactive-substrate assay for prostaglandin synthase (EC 1.14.99.1), which uses t.l.c. to measure simultaneously different prostaglandins synthesized from one precursor substrate, was developed. Rabbit kidney-medulla prostaglandin synthase catalyses the formation of prostaglandin E2, prostaglandin F2alpha and prostaglandin D2 from arachidonic acid. Fractionation of crude homogenates indicated that the microsomal fraction possessed the highest specific activity of prostaglandin synthase, whereas the soluble fraction exhibited little enzyme activity but rather contained a heat-labile inhibitory macromolecular factor(s), which might be attributed to the serum albumin present in this fraction. The microsomal fraction possessed low intrinsic enzyme activity, but the actvity could be fully stimulated by the presence of both GSH (reduced glutathione) and a phenolic cofactor. Only cysteine could partially replace GSH, whereas other thiols were inactive and some were even inhibitory. A variety of phenolic compounds, including catecholamines, dopamine (3,4-dihydroxyphenethylamine), 5-hydroxytryptamine and quinol, were active in stimulating prostaglandin synthase. In all cases, the stimulation was reflected in the synthesis of all three prostaglandins with ratios not significantly altered by different phenolic cofactors. The synthesis of each of the different prostaglandins appeared to have similar pH optima. The enzyme system was not inhibited by thiol-group inhibitors or a variety of metal chelators except for cyanide and 8-hydroxyquinoline. Characterization of the kidney-medulla prostaglandin synthase system indicated that it exhibited properties similar to those of the enzyme system present in seminal vesicles. | [
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PMID:7236 | Interactions of some acceptors with superoxide anion radicals formed by the NADPH-specific flavoprotein in rat liver microsomal fractions. | In rat liver microsomal fractions oxidation of adrenaline was effected by superoxide anion radicals (O2-), whereas cytochrome c, 2,6-dichlorophenol-indophenol and ferricyanide accepted electrons from NADPH-specific flavoprotein only directly. Nitro Blue Tetrazolium was reduced both by O2- and by the direct acceptance of electrons. Elevation of pH and addition of menadione shift the Nitro Blue Tetrazolium reduction towards the O2--dependent pathway. From the values of the kinetic constants for interaction of adrenaline and Nitro Blue Tetrazolium with NADPH-specific flavoprotein, the rates of generation of O2- in rat liver microsomal fraction were determined. | Interactions of some acceptors with superoxide anion radicals formed by the NADPH-specific flavoprotein in rat liver microsomal fractions. In rat liver microsomal fractions oxidation of adrenaline was effected by superoxide anion radicals (O2-), whereas cytochrome c, 2,6-dichlorophenol-indophenol and ferricyanide accepted electrons from NADPH-specific flavoprotein only directly. Nitro Blue Tetrazolium was reduced both by O2- and by the direct acceptance of electrons. Elevation of pH and addition of menadione shift the Nitro Blue Tetrazolium reduction towards the O2--dependent pathway. From the values of the kinetic constants for interaction of adrenaline and Nitro Blue Tetrazolium with NADPH-specific flavoprotein, the rates of generation of O2- in rat liver microsomal fraction were determined. | [
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PMID:7237 | L-lactate transport in Ehrlich ascites-tumour cells. | Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids. | L-lactate transport in Ehrlich ascites-tumour cells. Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids. | [
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PMID:7238 | Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase. | 6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase. | Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase. 6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase. | [
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PMID:7239 | Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315. | 1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment... | Interactions of the lanthanide- and hapten-binding sites in the Fv fragment from the myeloma protein MOPC 315. 1. The interactions of lanthanide metals and dinitrophenyl spin-label haptens with the Fv fragment of the mouse myeloma protein MOPC 315 were investigated by the techniques of fluorescence, e.s.r. (electron spin resonance) and high-resolution n.m.r. (nuclear magnetic resonance). 2. The protein fluorescence of Fv fragment at 340nm is quenched by the haptens (fluorescence enhancement, epsilon=0.15) and enhanced by Gd(III) (epsilon=1.14) and other lanthanides. The binding of the haptens studied here is insensitive to pH in the range 5.5-7.0 (dissociation constant KH=0.3-1.0 muM) and shows 1:1 stoicheiometry. The binding of Gd(III) also shows 1:1 stoicheiometry, but is pH-dependent; the binding constant (KM) varies from 10 muM at pH7.0 to 700 muM at pH4.8. La(III) binding is less sensitive to pH. The pH-dependences of the metal-binding constants imply that a group in the protein with pKa greater than or equal to 6.2 is involved in the binding, and probably also other groups with lower pKa values. 3. The apparent binding of the haptens is weakened about 20-fold by Gd(III), and vice versa. An equilibrium scheme involving a ternary complex with an interaction between the two binding sites is derived in Appendix I to explain the experimental results at two pH values. 4. Time-dependent fluorescence changes are observed in the presence of Gd(III) at pH5.5. A two-state kinetic scheme involving a 'slow' conformational change in the Fv fragment is derived in Appendix II to explain this time-dependence. This scheme is consistent with the antagonistic equilibrium behaviour. 5. The e.s.r. changes in the spin-label haptens on binding to Fv fragment and on the subsequent addition of lanthanides are consistent with the binding scheme for haptens and lanthanides proposed from the fluorescence studies. A difference between the limiting quenching of the e.s.r. signal from the bound haptens in the presence of saturating concentrations of Gd(III) and La(III) is attributed to dipolar interactions between bound Gd(III) and the nitroxide moiety of the bound hapten. The residual quenching with Gd(III) allows an estimate of 1.2nm to be made for the distance between the two paramagnetic centres. 6. The 270 MHz proton difference spectrum of the Fv fragment resulting from the addition of La(III) suggests that any metal-induced conformational changes are small and involve relatively few amino acid residues on the Fv fragment... | [
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PMID:7240 | Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement. | 1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments. | Isolation, by partial pepsin digestion, of the three collagen-like regions present in subcomponent Clq of the first component of human complement. 1. Digestion of human subcomponent C1q with pepsin at pH4.45 for 20h at 37 degrees C fragmented most of the non-collagen-like amino acid sequences in the molecule to small peptides, whereas the entire regions of collagen-like sequence that comprised 38% by weight of the subcomponent C1q were left intact. 2. The collagen-like fraction of the digest was eluted in the void volume of a Sephadex G-200 column, was was showm to be composed of two major fragments when examined by electrophoresis on polyacrylamide gels run in buffers containing sodium dodecyl sulphate. These fragments were separated on CM-cellulose at pH4.9 in buffers containing 7.5M-urea. 3. Human subcomponent C1q on reduction and alkylation yields equimolar amounnts of three chains, which have been designated A, B and C [Reid et al. (1972) Biochem. J. 130, 749-763]. One of the pepsin fragments was shown to be composed of the N-terminal 95 residues of the A chain linked, via residue A4, by a single disulphide bond to a residue in the sequence B2-B6 in the N-terminal 91 residues of the B chain. The second pepsin fragment was shown to be composed of a disulphide-linked dimer of the N-terminal 94 residues of the C chain, the only disulphide bond being located at residue C4.4. The mol. wts. of the unoxidized and oxidized pepsin fragments were estimated from their amino acid compositions to be 20 000 and 18 200 for the A-B and C-C dimers and 11 400, 8800 and 9600 for the collagen-like fragments of the A, B and C chains respectively. Estimation of the molecular weights of the peptic fragments by polyacrylamide-gel electrophoresis run in the presence of sodium dodecyl sulphate gave values that were approx. 50% higher than expected from the amino acid sequence data. This is probably due to the high collagen-like sequence content of these fragments. | [
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PMID:7241 | PH-dependence of the steady-state rate of a two-step enzymic reaction. | 1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated. | PH-dependence of the steady-state rate of a two-step enzymic reaction. 1. The pH-dependence is considered of a reaction between E and S that proceeds through an intermediate ES under "Briggs-Haldane' conditions, i.e. there is a steady state in ES and [S]o greater than [E]T, where [S]o is the initial concentration of S and [E]T is the total concentration of all forms of E. Reactants and intermediates are assumed to interconvert in three protonic states (E equilibrium ES; EH equilibrium EHS; EH2 equilibrium EH2S), but only EHS provides products by an irreversible reaction whose rate constant is kcat. Protonations are assumed to be so fast that they are all at equilibrium. 2. The rate equation for this model is shown to be v = d[P]/dt = (kcat.[E]T[S]o/A)/[(KmBC/DA) + [S]o], where Km is the usual assembly of rate constants around EHS and A-D are functions of the form (1 + [H]/K1 + K2/[H]), in which K1 and K2 are: in A, the molecular ionization constants of ES; in B, the analogous constants of E; in C and D, apparent ionization constants composed of molecular ionization constants (of E or ES) and assemblies of rate constants. 3. As in earlier treatments of this type of reaction which involve either the assumption that the reactants and intermediate are in equilibrium or the assumption of Peller & Alberty [(1959) J. Am. Chem. Soc. 81, 5907-5914] that only EH and EHS interconvert directly, the pH-dependence of kcat. is determined only by A. 4. The pH-dependence of Km is determined in general by B-C/A-D, but when reactants and intermediate are in equilibrium, C identical to D and this expression simplifies to B/A. 5. The pH-dependence of kcat./Km, i.e. of the rate when [S]o less than Km, is not necessarily a simple bell-shaped curve characterized only by the ionization constants of B, but is a complex curve characterized by D/B-C. 6. Various situations are discussed in which the pH-dependence of kcat./Km is determined by assemblies simpler than D/B-C. The special situation in which a kcat./Km-pH profile provides the molecular pKa values of the intermediate ES complex is delineated. | [
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PMID:7242 | Some properties of a microsomal oleate desaturase from leaves. | 1. When [1-14C]oleoyl-CoA was incubated with a pea-leaf homogenate oleate was both incorporated into microsomal 3-sn-phosphatidylcholine and released as the unesterified fatty acid. The proportion of oleate incorporated into this phospholipid was dependent on the relative amounts of thiol ester and microsomal preparation present in reactions. 2. At the concentrations of microsomal preparation and [14C]oleoyl-CoA used to study oleate desaturation the metabolism of the thiol ester was essentially complete after 5 min incubation, but the loss of label from 3-sn-phosphatidylcholine oleate and the concomitant increase in radioactivity in the linoleate of this phospholipid proceeded at approximately linear rates over a 60 min period. The kinetics of labelling of unesterified linoleate was consistent with the view that this labelled fatty acid was derived from 3-sn-phosphatidylcholine. 3. Oleate desaturation required oxygen and with unwashed microsomal fractions was stimulated either by NADPH or by the 105 000g supernatant. Washed microsomal preparations did not catalyse desaturation, but actively was restored by the addition of NADPH, 105 000G supernatant or Sephadex-treated supernatant. NADPH could be replaced by NADH or NADP+, but not by NAD+. 4. Microsomal fractions from mature and immature maize lamina and expanding spinach leaves also rapidly incorporated oleate from ([14C]oleoyl-CoA into 3-sn-phosphatidylcholine, but desaturation of 3-sn-phosphatidylcholine oleate was detected only with microsomal preparations from immature maize lamina. 5. It is proposed that leaf microsomal preparations posses an oleate desaturase for which 3-sn-phosphatidylcholine oleate is either the substrate or an immediate precursor of the substrate. | Some properties of a microsomal oleate desaturase from leaves. 1. When [1-14C]oleoyl-CoA was incubated with a pea-leaf homogenate oleate was both incorporated into microsomal 3-sn-phosphatidylcholine and released as the unesterified fatty acid. The proportion of oleate incorporated into this phospholipid was dependent on the relative amounts of thiol ester and microsomal preparation present in reactions. 2. At the concentrations of microsomal preparation and [14C]oleoyl-CoA used to study oleate desaturation the metabolism of the thiol ester was essentially complete after 5 min incubation, but the loss of label from 3-sn-phosphatidylcholine oleate and the concomitant increase in radioactivity in the linoleate of this phospholipid proceeded at approximately linear rates over a 60 min period. The kinetics of labelling of unesterified linoleate was consistent with the view that this labelled fatty acid was derived from 3-sn-phosphatidylcholine. 3. Oleate desaturation required oxygen and with unwashed microsomal fractions was stimulated either by NADPH or by the 105 000g supernatant. Washed microsomal preparations did not catalyse desaturation, but actively was restored by the addition of NADPH, 105 000G supernatant or Sephadex-treated supernatant. NADPH could be replaced by NADH or NADP+, but not by NAD+. 4. Microsomal fractions from mature and immature maize lamina and expanding spinach leaves also rapidly incorporated oleate from ([14C]oleoyl-CoA into 3-sn-phosphatidylcholine, but desaturation of 3-sn-phosphatidylcholine oleate was detected only with microsomal preparations from immature maize lamina. 5. It is proposed that leaf microsomal preparations posses an oleate desaturase for which 3-sn-phosphatidylcholine oleate is either the substrate or an immediate precursor of the substrate. | [
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PMID:7243 | Isolation and properties of alpha-D-mannosidase from human kidney. | Alpha-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, alpha-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl alpha-D-mannoside as substrate. The purified alpha-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 X 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and gamma-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, alpha-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C. | Isolation and properties of alpha-D-mannosidase from human kidney. Alpha-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, alpha-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl alpha-D-mannoside as substrate. The purified alpha-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 X 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and gamma-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, alpha-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C. | [
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PMID:7244 | Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G. | 1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms. | Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G. 1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms. | [
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PMID:7245 | Human cathepsin G. Catalytic and immunological properties. | 1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes. | Human cathepsin G. Catalytic and immunological properties. 1. The specificity of cathepsin G, a neutral proteinase from human spleen, was examined by use of low-molecular-weight substrates. The enzyme was found to hydrolyse several synthetic substrates also hydrolysed by chymotrypsin, but with different kinetic constants. 2. Maximal activity against benzoyl-DL-phenylalanine 2-naphthol ester and azo-casein was in the range pH 7.5-8.0. 3. The sensitivity of cathepsin G to the action of potential inhibitors was determined, and compared with those of bovine chymotrypsin and subtilisin. Cathepsin G showed the characteristics of a serine proteinase, but was less affected by the chloromethyl ketone of tosylphenylalanine than was chymotrypsin. 4. A rabbit anti-(human cathepsin G) serum was raised, and precipitin lines formed in agarose gel were stained for activity of the enzyme. 5. Cathepsin G was shown to be immunologically identical with the chymotrypsin-like enzyme of the azurophil granules of the neutrophil granulocytes. | [
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PMID:7246 | Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids. | 1. The surface potentials of mixed monolayers of synthetic phospholipids with lipids that are fusogenic for hen erythrocytes were investigated. 2. At pH 5.6 and 10, but not at pH2, mixed monolayers of the fusogenic lipid, glycerol mono-oleate, with phosphatidylcholine exhibited negative deviations from the ideality rule in surface potential per molecule which were accompanied by negative deviations in mean molecular area. 3. Interactions of this type were not seen with chemically related but non-fusogenic lipids, nor were they found in mixed monolayers of any of the lipids with phosphatidylethanolamine. 4. Experiments with dihexadecyl phosphate and hexadecyltrimethyl-ammonium indicated that the complete head group of phosphatidylcholine is required for its observed behaviour with fusogenic lipids. 5. Bivalent cations (Ca2+, UO2(2+) or Zn2+) in the subphase at pH 5.6 significantly modified the behaviour of mixed monolayers of fusogenic lipids with phospholipids; there was a parallel perturbing effect of fusogenic lipids on interactions between monolayers of phospholipids and bivalent cations. 6. Possible molecular interactions of fusogenic lipids with membrane phospholipids, and the role of Ca2+, are discussed which may be relevant to cell fusion in erythrocytes induced by low-melting lipids in the presence of Ca2+. | Polar-group behaviour in mixed monolayers of phospholipids and fusogenic lipids. 1. The surface potentials of mixed monolayers of synthetic phospholipids with lipids that are fusogenic for hen erythrocytes were investigated. 2. At pH 5.6 and 10, but not at pH2, mixed monolayers of the fusogenic lipid, glycerol mono-oleate, with phosphatidylcholine exhibited negative deviations from the ideality rule in surface potential per molecule which were accompanied by negative deviations in mean molecular area. 3. Interactions of this type were not seen with chemically related but non-fusogenic lipids, nor were they found in mixed monolayers of any of the lipids with phosphatidylethanolamine. 4. Experiments with dihexadecyl phosphate and hexadecyltrimethyl-ammonium indicated that the complete head group of phosphatidylcholine is required for its observed behaviour with fusogenic lipids. 5. Bivalent cations (Ca2+, UO2(2+) or Zn2+) in the subphase at pH 5.6 significantly modified the behaviour of mixed monolayers of fusogenic lipids with phospholipids; there was a parallel perturbing effect of fusogenic lipids on interactions between monolayers of phospholipids and bivalent cations. 6. Possible molecular interactions of fusogenic lipids with membrane phospholipids, and the role of Ca2+, are discussed which may be relevant to cell fusion in erythrocytes induced by low-melting lipids in the presence of Ca2+. | [
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PMID:7247 | Phosphonomethyl analogues of hexose phosphates. | The analogue of fructose 1,6-bisphosphate in which the phosphate group, -O-PO3H2, on C-6 is replaced by the phosphonomethyl group, -CH2-PO3H2, was made enzymically from the corresponding analogue of 3-phosphoglycerate. It was a substrate for aldolase, which was used to form it, but not for fructose 1,6-bisphosphatase. It was hydrolysed chemically to yield the corresponding analogue of fructose 6-phosphate [i.e. 6-deoxy-6-(phosphonomethyl)-D-fructose, or, more strictly, 6,7-dideoxy-7-phosphono-D-arabino-2-heptulose]. This proved to be a substrate for the sequential actions of glucose 6-phosphate isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Thus seven out of the nine enzymes of the glycolytic and pentose phosphate pathways so far tested catalyse the reactions of the phosphonomethyl isosteres of their substrates. | Phosphonomethyl analogues of hexose phosphates. The analogue of fructose 1,6-bisphosphate in which the phosphate group, -O-PO3H2, on C-6 is replaced by the phosphonomethyl group, -CH2-PO3H2, was made enzymically from the corresponding analogue of 3-phosphoglycerate. It was a substrate for aldolase, which was used to form it, but not for fructose 1,6-bisphosphatase. It was hydrolysed chemically to yield the corresponding analogue of fructose 6-phosphate [i.e. 6-deoxy-6-(phosphonomethyl)-D-fructose, or, more strictly, 6,7-dideoxy-7-phosphono-D-arabino-2-heptulose]. This proved to be a substrate for the sequential actions of glucose 6-phosphate isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Thus seven out of the nine enzymes of the glycolytic and pentose phosphate pathways so far tested catalyse the reactions of the phosphonomethyl isosteres of their substrates. | [
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PMID:7275 | A metal complexing property of furosemide and bumetanide: determination of pK and stability constant. | Furosemide and bumetanide have been found to have different pK values and undergo complex formation with several metal ions in dioxane-water solution. pK values of these diuretics were determined. Evidence for complex formation was provided by a drop in pH during complex formation, production of a characteristic color and precipitation. | A metal complexing property of furosemide and bumetanide: determination of pK and stability constant. Furosemide and bumetanide have been found to have different pK values and undergo complex formation with several metal ions in dioxane-water solution. pK values of these diuretics were determined. Evidence for complex formation was provided by a drop in pH during complex formation, production of a characteristic color and precipitation. | [
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PMID:7276 | [Drug-drug interactions (author's transl)]. | This short outline of drug-drug interactions does not claim to cover the entire field. The task of this paper is to illustrate the most important principles of drug-drug interactions by paradigms taken from the experience of the practitioner. One consequence of drug-drug interactions is the change in pharmacolinetic parameters important for the therapeutical effect of drugs in the organism. Very often the elucidation of the mechanisms of drug-drug interactions in man is impossible; therefore, for clinical pharmacologists experiments on animals remain the tool in order to gain more knowledge in this field. | [Drug-drug interactions (author's transl)]. This short outline of drug-drug interactions does not claim to cover the entire field. The task of this paper is to illustrate the most important principles of drug-drug interactions by paradigms taken from the experience of the practitioner. One consequence of drug-drug interactions is the change in pharmacolinetic parameters important for the therapeutical effect of drugs in the organism. Very often the elucidation of the mechanisms of drug-drug interactions in man is impossible; therefore, for clinical pharmacologists experiments on animals remain the tool in order to gain more knowledge in this field. | [
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PMID:7277 | [Galenical possibilities and problems in protraction of drug effects (author's transl)]. | In recent years, dosage forms with sustained release have obtained a significant importance. The technological possibilities for manufacturing are described as coating, embedding and matrix procedures. The range of auxiliary substances, which are responsible for the retardation of drug activity, reaches from lipophilic compounds as lipoids, fatty alcohols and compounds which are forming hydrogels as cellulose derivatives and natural polysaccharides to synthetic polymers derived from acrylic acid. The formulation of the dosage forms requires particular care in respect to the amount of initial and maintenance doses. On account of technological processes, for example during manufacturing of tablets, under certain circumstances the liberation rate is altered. In vitro test methods allow comparisons only then when the results can be counter-checked by in vitro experiments. The release of drug follows different mechanisms, which are described, entirely or in part, to be reactions following the time law of zero or first order. In special cases, a linear correlation is observed as a function of square root of time. The calculation of given special equations for events within the dosage form is feasible from blood-level values. | [Galenical possibilities and problems in protraction of drug effects (author's transl)]. In recent years, dosage forms with sustained release have obtained a significant importance. The technological possibilities for manufacturing are described as coating, embedding and matrix procedures. The range of auxiliary substances, which are responsible for the retardation of drug activity, reaches from lipophilic compounds as lipoids, fatty alcohols and compounds which are forming hydrogels as cellulose derivatives and natural polysaccharides to synthetic polymers derived from acrylic acid. The formulation of the dosage forms requires particular care in respect to the amount of initial and maintenance doses. On account of technological processes, for example during manufacturing of tablets, under certain circumstances the liberation rate is altered. In vitro test methods allow comparisons only then when the results can be counter-checked by in vitro experiments. The release of drug follows different mechanisms, which are described, entirely or in part, to be reactions following the time law of zero or first order. In special cases, a linear correlation is observed as a function of square root of time. The calculation of given special equations for events within the dosage form is feasible from blood-level values. | [
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PMID:7278 | [Metabolic degradation of cartilage by leucocyte enzymes under the influence of antirheumatic drugs (author's transl)]. | Lately the importance and participation of leucocytes in rheumatoid arthritis have been discussed. In order to study catabolic reactions in connective tissue we investigated the autolytic liberation of mucopolysaccharides in cartilage alone and in the presence of leucocyte enzymes. Factors of optimal experimental conditions, such as pH and constituents of incubation medium, incubation time and the number of leucocytes, were determined. In further experiments we studied the influence of antirheumatic drugs, such as sodium salicylate, phenylbutazone, pentosanpoly-sulfate and gold thiopolypeptide, on cartilage degradation. In our experiments only phenylbutazone and pentosanpoly-sulfate exerted an inhibitory effect on leucocyte-stimulated degradation of cartilage. The relevance of this finding for the therapeutic use of these drugs is evaluated. | [Metabolic degradation of cartilage by leucocyte enzymes under the influence of antirheumatic drugs (author's transl)]. Lately the importance and participation of leucocytes in rheumatoid arthritis have been discussed. In order to study catabolic reactions in connective tissue we investigated the autolytic liberation of mucopolysaccharides in cartilage alone and in the presence of leucocyte enzymes. Factors of optimal experimental conditions, such as pH and constituents of incubation medium, incubation time and the number of leucocytes, were determined. In further experiments we studied the influence of antirheumatic drugs, such as sodium salicylate, phenylbutazone, pentosanpoly-sulfate and gold thiopolypeptide, on cartilage degradation. In our experiments only phenylbutazone and pentosanpoly-sulfate exerted an inhibitory effect on leucocyte-stimulated degradation of cartilage. The relevance of this finding for the therapeutic use of these drugs is evaluated. | [
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PMID:7279 | [MASCA-model of biochemical-pharmacological drug research/Part VIII: Examples (author's transl)]. | In this report two examples (inhibition of alcohol dehydrogenase by pyridine and benzamide derivates) are given for the interpretation of the MASCA-model. | [MASCA-model of biochemical-pharmacological drug research/Part VIII: Examples (author's transl)]. In this report two examples (inhibition of alcohol dehydrogenase by pyridine and benzamide derivates) are given for the interpretation of the MASCA-model. | [
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PMID:7280 | [In vitro inhibition of oxidative N-demethylation with carbon disulfide]. | Earlier findings have shown that in experimental animals (rat) and in man inhaled carbon disulphide (CS2) reversibly inhibits the non-specific oxidative drug metabolism caused by hepatic microsomal enzymes. Very little is known concerning the underlying mechanism. The present investigations were undertaken to throw light on this question. After addition of an NADPH-regenerating system to liver microsomes isolated from adult female Wistar rats, the oxidative N-demethylation of aminopyrine was measured under simultaneous exposure to CS2 by quantitatively determining the formaldehyde obtained; the resulting data were evaluated using enzyme-kinetic parameters according to Lineweaver-Burk: 1. Following acute exposure to low and medium-grade CS2 concentrations (20-400 ppm/8 h), the pattern of inhibition in rat liver microsomes is identical to that obtained in normal liver microsomes to which CS2 had been added. This finding seems to suggest that the inhibitory process under in vivo and in vitro conditions is based on one and the same molecular mechanism. 2. Upon addition of CS2 the in vitro pattern of inhibition corresponds to a strong mixed-type inhibition. 3. It is concluded from the enzyme-kinetic behaviour that CS2 attacks at two different sites of the enzyme molecule: Binding to the first site is followed by inhibition, as evidenced by the rise of Km; after saturation of this site, a second site is occupied resulting in a reactivation and, beyond this, an activation of enzyme output, as shown by the decrease of Km. CS2 exhibits a high affinity for the first site, and a low affinity for the second site. Binding at the first site is reversible. The possibility that the active centre in the enzyme molecule is the site where binding of the inhibitor occurs is ruled out. 4. A continuous decrease of Vmax at increasing inhibitor concentration is causally related with the formation of an enzyme/substrate inhibitor complex. 5. The CS2 added to the microsomes can be eliminated by helium gas; this is followed by the return of the original enzyme activity. It is concluded from this behaviour that under in vitro conditions CS2 itself (rather than its metabolites) acts as the inhibitor. 6. Oxygen treatment of the microsome-containing reaction mixture enhances the inhibition. In substrate-free control mixtures, addition of CS2 was followed by the dose-dependent formation of formaldehyde; a causal explanation is not readily available at this time. | [In vitro inhibition of oxidative N-demethylation with carbon disulfide]. Earlier findings have shown that in experimental animals (rat) and in man inhaled carbon disulphide (CS2) reversibly inhibits the non-specific oxidative drug metabolism caused by hepatic microsomal enzymes. Very little is known concerning the underlying mechanism. The present investigations were undertaken to throw light on this question. After addition of an NADPH-regenerating system to liver microsomes isolated from adult female Wistar rats, the oxidative N-demethylation of aminopyrine was measured under simultaneous exposure to CS2 by quantitatively determining the formaldehyde obtained; the resulting data were evaluated using enzyme-kinetic parameters according to Lineweaver-Burk: 1. Following acute exposure to low and medium-grade CS2 concentrations (20-400 ppm/8 h), the pattern of inhibition in rat liver microsomes is identical to that obtained in normal liver microsomes to which CS2 had been added. This finding seems to suggest that the inhibitory process under in vivo and in vitro conditions is based on one and the same molecular mechanism. 2. Upon addition of CS2 the in vitro pattern of inhibition corresponds to a strong mixed-type inhibition. 3. It is concluded from the enzyme-kinetic behaviour that CS2 attacks at two different sites of the enzyme molecule: Binding to the first site is followed by inhibition, as evidenced by the rise of Km; after saturation of this site, a second site is occupied resulting in a reactivation and, beyond this, an activation of enzyme output, as shown by the decrease of Km. CS2 exhibits a high affinity for the first site, and a low affinity for the second site. Binding at the first site is reversible. The possibility that the active centre in the enzyme molecule is the site where binding of the inhibitor occurs is ruled out. 4. A continuous decrease of Vmax at increasing inhibitor concentration is causally related with the formation of an enzyme/substrate inhibitor complex. 5. The CS2 added to the microsomes can be eliminated by helium gas; this is followed by the return of the original enzyme activity. It is concluded from this behaviour that under in vitro conditions CS2 itself (rather than its metabolites) acts as the inhibitor. 6. Oxygen treatment of the microsome-containing reaction mixture enhances the inhibition. In substrate-free control mixtures, addition of CS2 was followed by the dose-dependent formation of formaldehyde; a causal explanation is not readily available at this time. | [
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PMID:7281 | [The influence of neuroleptic drugs on urinary excretion of non-protein nitrogen (author's transl)]. | During treatment with thioxanthenes or phenothiazines of schizophrenic patients non-protein nitrogen in urine was measured. The values were calculated in relation to the excretion of creatinine. a) Flupentixol or fluphenazine applied in optimal dosage, increased the excretion of urea and the amino acids asp, glu + gln, and gly. b) Moreover, if the drug induced a parkinsonoid (thioridazine) the excretion of ser and thr was increased, too. The usual desalting procedure by ion-exchanging resins before chromatography increases the contents of several amino acids, e.g. asp, asn, ala, gly, cys, ser, thr, indicating a breakdown of some instable products. | [The influence of neuroleptic drugs on urinary excretion of non-protein nitrogen (author's transl)]. During treatment with thioxanthenes or phenothiazines of schizophrenic patients non-protein nitrogen in urine was measured. The values were calculated in relation to the excretion of creatinine. a) Flupentixol or fluphenazine applied in optimal dosage, increased the excretion of urea and the amino acids asp, glu + gln, and gly. b) Moreover, if the drug induced a parkinsonoid (thioridazine) the excretion of ser and thr was increased, too. The usual desalting procedure by ion-exchanging resins before chromatography increases the contents of several amino acids, e.g. asp, asn, ala, gly, cys, ser, thr, indicating a breakdown of some instable products. | [
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PMID:7286 | Gastric secretion and fermentation in the suckling pig. | 1. The contribution to acidification of the stomach contents of pigs by hydrochloric acid secretion or by lactic acid produced by fermentation was studied in fifteen suckling pigs from six litters born and reared either in a 'conventional' environment or in an isolated 'clean' environment. Sequential samples of stomach contents obtained during periods of up to 24 h were analysed for their chloride and lactic acid contents, pH and total titratable acidity. These values gave a measure of organic and inorganic acids respectively. 2. Six pigs from two litters born and reared in a 'clean' environment had acid secretion in the stomach at 2 d of age, and the concentrations of lactic acid in stomach contents remained low (0-40 mmol/l) throughout the suckling period. 3. Eight pigs from three litters born and reared in a 'conventional' environment, and a ninth pig born in this environment but moved to the 'clean' environment at 24 h of age, had lactic acid in concentrations of up to 250 mmol/l in stomach contents within the 1st week of life. The pattern of lactic acid production (and hence the acidity of stomach contents) was governed by frequency of suckling. 4. Both between- and within-litter variation in the age of onset of HC1 secretion was evident in the group reared in a 'conventional' environment, and when HC1 secretion did occur it was usually accompanied by a reduction in lactic acid production. 5. It is concluded: (1) that the environment at birth is important in determining the fermentative ability of the stomach flora; (2) that if lactic acid is produced in large amounts in the stomach, it may partly or completely inhibit acidification by HC1. | Gastric secretion and fermentation in the suckling pig. 1. The contribution to acidification of the stomach contents of pigs by hydrochloric acid secretion or by lactic acid produced by fermentation was studied in fifteen suckling pigs from six litters born and reared either in a 'conventional' environment or in an isolated 'clean' environment. Sequential samples of stomach contents obtained during periods of up to 24 h were analysed for their chloride and lactic acid contents, pH and total titratable acidity. These values gave a measure of organic and inorganic acids respectively. 2. Six pigs from two litters born and reared in a 'clean' environment had acid secretion in the stomach at 2 d of age, and the concentrations of lactic acid in stomach contents remained low (0-40 mmol/l) throughout the suckling period. 3. Eight pigs from three litters born and reared in a 'conventional' environment, and a ninth pig born in this environment but moved to the 'clean' environment at 24 h of age, had lactic acid in concentrations of up to 250 mmol/l in stomach contents within the 1st week of life. The pattern of lactic acid production (and hence the acidity of stomach contents) was governed by frequency of suckling. 4. Both between- and within-litter variation in the age of onset of HC1 secretion was evident in the group reared in a 'conventional' environment, and when HC1 secretion did occur it was usually accompanied by a reduction in lactic acid production. 5. It is concluded: (1) that the environment at birth is important in determining the fermentative ability of the stomach flora; (2) that if lactic acid is produced in large amounts in the stomach, it may partly or completely inhibit acidification by HC1. | [
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PMID:7287 | Broad-line nuclear magnetic resonance studies of chloroperoxidase. | Chloroperoxidase, a heme glycoprotein isolated from the mold Caldariomyces fumago, was studied by NMR relaxation techniques. Interaction of the chloride ion substrate with the enzyme may be analyzed as consisting of at least three contributions: a weak interaction with the iron atom, nonspecific anion-protein interactions, and a specific interaction generated at low pH. The data indicate that a specific interaction, which develops in parallel with enzyme activity at low pH, does not occur at the iron atom first coordination sphere site. The results are summarized in terms of an enzymatic mechanism not involving chloride ion coordination to the iron atom. | Broad-line nuclear magnetic resonance studies of chloroperoxidase. Chloroperoxidase, a heme glycoprotein isolated from the mold Caldariomyces fumago, was studied by NMR relaxation techniques. Interaction of the chloride ion substrate with the enzyme may be analyzed as consisting of at least three contributions: a weak interaction with the iron atom, nonspecific anion-protein interactions, and a specific interaction generated at low pH. The data indicate that a specific interaction, which develops in parallel with enzyme activity at low pH, does not occur at the iron atom first coordination sphere site. The results are summarized in terms of an enzymatic mechanism not involving chloride ion coordination to the iron atom. | [
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PMID:7288 | Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide. | Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides. | Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide. Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides. | [
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PMID:7289 | Effects of substrate and inhibitor binding on thermal and proteolytic inactivation of rat liver transhydrogenase. | The thermostability and proteolytic inactivation of rat liver submitochondrial particle transhydrogenase was studied in the presence of pyridine dinucleotide substrates and a variety of divalent metal and nucleotide inhibitors. Relative to the unliganded enzyme, the NADPH-enzyme complex was more thermostable and showed a twofold greater rate of tryptic inactivation, while the NADP+-enzyme complex was more thermolabile and only slightly more susceptible to tryptic inactivation. Neither NAD+ nor NADH significantly affected thermostability or proteolysis. Similar effects of these ligands were observed for the non-energy-linked and energy-linked transhydrogenase reactions, indicating that both activities are catalyzed by the same enzyme. In thermal experiments, acetyl-CoA, 2'-AMP, and NMNH stabilized, palmitoyl-CoAlabilized, and dephospho-CoA, CoA, NMN+, and 5'-AMP had little effect on enzyme stability. Tryptic inactivation was inhibited by 2'-AMP and NMN+ but was not influenced by the other nucleotide inhibitors. Divalent metal ion inhibitors (Mg2+, Ca2+, Mn2+, Ba2+, and Sr2+) stabilized transhydrogenase against thermal inactivation and promoted tryptic inactivation. | Effects of substrate and inhibitor binding on thermal and proteolytic inactivation of rat liver transhydrogenase. The thermostability and proteolytic inactivation of rat liver submitochondrial particle transhydrogenase was studied in the presence of pyridine dinucleotide substrates and a variety of divalent metal and nucleotide inhibitors. Relative to the unliganded enzyme, the NADPH-enzyme complex was more thermostable and showed a twofold greater rate of tryptic inactivation, while the NADP+-enzyme complex was more thermolabile and only slightly more susceptible to tryptic inactivation. Neither NAD+ nor NADH significantly affected thermostability or proteolysis. Similar effects of these ligands were observed for the non-energy-linked and energy-linked transhydrogenase reactions, indicating that both activities are catalyzed by the same enzyme. In thermal experiments, acetyl-CoA, 2'-AMP, and NMNH stabilized, palmitoyl-CoAlabilized, and dephospho-CoA, CoA, NMN+, and 5'-AMP had little effect on enzyme stability. Tryptic inactivation was inhibited by 2'-AMP and NMN+ but was not influenced by the other nucleotide inhibitors. Divalent metal ion inhibitors (Mg2+, Ca2+, Mn2+, Ba2+, and Sr2+) stabilized transhydrogenase against thermal inactivation and promoted tryptic inactivation. | [
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PMID:7290 | N-(1-pyrene)maleimide: a fluorescent cross-linking reagent. | N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins. | N-(1-pyrene)maleimide: a fluorescent cross-linking reagent. N-(1-Pyrene)maleimide is nonfluorescent in aqueous solution but forms strongly fluorescent adducts with sulfhydryl groups of organic compounds or proteins. The conjugation reactions of N-(1-pyrene)maleimide are relatively fast and can be monitored by the increase in fluorescence intensity of the pyrene chromophore. In cases where primary amino groups are also present in the system, we have observed a red shift of the emission spectra of the fluorescent adducts subsequent to the initial conjugation, as characterized by the disappearance of three emission peaks at 376, 396, and 416 nm, and the appearance of two new peaks at 386 and 405 nm. Model studies with N-(1-pyrene)maleimide adducts of L-cysteine and cysteamine indicate that the spectral shift is the result of an intramolecular aminolysis of the succinimido ring in the adducts. Evidence from both chemical analysis and nuclear magnetic resonance studies of the addition products supports this reaction scheme. N-(1-Pyrene)maleimide adducts of N-acetyl-L-cysteine and beta-mercaptoethanol, which have no free amino group, do not exhibit a spectral shift. Among several protein conjugates only the N-(1-pyrene)maleimide adduct of bovine serum albumin (PM-BSA) shows the spectral shift resembling that of PM-cysteine. N-(1-Pyrene)maleimide reacts with the sulfhydryl group of the single cysteine residue at position 34 in BSA. The finding that the alpha-amino group of the N-terminus in PM-BSA is blocked after the spectral shift is completed strongly suggests that N-(1-pyrene)maleimide cross-links the N-terminus and the cysteine residue in BSA. The relative proximity of the sulfhydryl and amino groups is very critical in the cross-linking as demonstrated by the observation that the spectral shift observed with PM-BSA can be prevented by addition of denaturing reagents such as 1% sodium dodecyl sulfate immediately after labeling, and by the failure of PM-glutathione to undergo the intramolecular aminolysis. Since the intramolecular rearrangement of PM adducts is associated with characteristic fluorescence changes, N-(1-pyrene)maleimide can serve as a fluorescent cross-linking reagent which provides information about the spatial proximity of sulfhydryl and amino groups in proteins. | [
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PMID:7291 | Analysis of the interaction of organic phosphates with hemoglobin. | The interaction of organic phosphates with hemoglobin is studied by use of a simple thermodynamic approach. A model-independent analysis is employed to evaluate the accuracy of Adair constants determined in the presence of 2,3-diphosphoglycerate (DPG). The change of oxygen affinity in the presence of phosphates is related to the macroscopic phosphate binding constants of oxy- and deoxyhemoglobin and used to extract such binding constants from oxygen equilibrium measurements. The change of the Bohr effect in the presence of phosphates and the competitive binding of carbon dioxide and DPG are treated quantitatively. The binding of organic phosphates is incorporated into an allosteric model, in which the effect of phosphate on both tertiary and quaternary structure changes is included. By use of this model, the factors which can be responsible for the increased functional heterogeneity of alpha and beta chains in the presence of phosphates are clarified. | Analysis of the interaction of organic phosphates with hemoglobin. The interaction of organic phosphates with hemoglobin is studied by use of a simple thermodynamic approach. A model-independent analysis is employed to evaluate the accuracy of Adair constants determined in the presence of 2,3-diphosphoglycerate (DPG). The change of oxygen affinity in the presence of phosphates is related to the macroscopic phosphate binding constants of oxy- and deoxyhemoglobin and used to extract such binding constants from oxygen equilibrium measurements. The change of the Bohr effect in the presence of phosphates and the competitive binding of carbon dioxide and DPG are treated quantitatively. The binding of organic phosphates is incorporated into an allosteric model, in which the effect of phosphate on both tertiary and quaternary structure changes is included. By use of this model, the factors which can be responsible for the increased functional heterogeneity of alpha and beta chains in the presence of phosphates are clarified. | [
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PMID:7292 | 15N nuclear magnetic resonance of flavins. | Ninety-nine percent 15N-enriched flavins were synthesized and their proton decoupled 15N resonances were observed. The enriched compounds were [1,3-15N]riboflavin, [1,3,5-15N]riboflavin, [1,3-15N]riboflavin 5'-phosphate, [1,3,5-15N]riboflavin 5'-phosphate, and [1,3,5-15N] flavin adenine dinucleotide, [1,3,5-15N] lumiflavin, and [1,3,5-15N] lumichrome. By comparison of their spectra and from th- nuclear Overhauser effect data each 15N resonance peak could be assigned to each 15N nucleus. The order of the chemical shifts well corresponds to that of the calculated pi-electron densities. The N-3 nucleus gives the most intense inverted peak and the N-5 nucleus a small noninverted peak. By changing pH from neutral to alkaline, the chemical shift and the intensity of signal were mostly affected in the N-3 resonance of riboflavin 5'-phosphate. The N-5 signal of flavin adenine dinucleotide showed a fairly large downfield shift with the increase of temperature. These observations can be well interpreted by the chemical structure and the proposed conformation of riboflavin 5'-phosphate and flavin adenine dinucleotide. | 15N nuclear magnetic resonance of flavins. Ninety-nine percent 15N-enriched flavins were synthesized and their proton decoupled 15N resonances were observed. The enriched compounds were [1,3-15N]riboflavin, [1,3,5-15N]riboflavin, [1,3-15N]riboflavin 5'-phosphate, [1,3,5-15N]riboflavin 5'-phosphate, and [1,3,5-15N] flavin adenine dinucleotide, [1,3,5-15N] lumiflavin, and [1,3,5-15N] lumichrome. By comparison of their spectra and from th- nuclear Overhauser effect data each 15N resonance peak could be assigned to each 15N nucleus. The order of the chemical shifts well corresponds to that of the calculated pi-electron densities. The N-3 nucleus gives the most intense inverted peak and the N-5 nucleus a small noninverted peak. By changing pH from neutral to alkaline, the chemical shift and the intensity of signal were mostly affected in the N-3 resonance of riboflavin 5'-phosphate. The N-5 signal of flavin adenine dinucleotide showed a fairly large downfield shift with the increase of temperature. These observations can be well interpreted by the chemical structure and the proposed conformation of riboflavin 5'-phosphate and flavin adenine dinucleotide. | [
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PMID:7293 | Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions. | Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate. | Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions. Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate. | [
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PMID:7294 | NADP+ and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation. | A mild oxidative stimulation of the hexose monophosphate pathway of human glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-deficient erythrocytes (Mediterranean variant) causes a significant drop in NADPH. These results, other than to confirm that glucose-6-phosphate dehydrogenase deficiency is a product deficiency disorder, demonstrate that under oxidative stimulation glutathione reductase may become functionally impaired and GSSG cannot be reduced at a sufficient rate. | NADP+ and NADPH in glucose-6-phosphate dehydrogenase-deficient erythrocytes under oxidative stimulation. A mild oxidative stimulation of the hexose monophosphate pathway of human glucose-6-phosphate dehydrogenase (EC 1.1.1.49)-deficient erythrocytes (Mediterranean variant) causes a significant drop in NADPH. These results, other than to confirm that glucose-6-phosphate dehydrogenase deficiency is a product deficiency disorder, demonstrate that under oxidative stimulation glutathione reductase may become functionally impaired and GSSG cannot be reduced at a sufficient rate. | [
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PMID:7295 | Purification and properties of ATPase inhibitor from rat liver mitochondria. | (1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered. | Purification and properties of ATPase inhibitor from rat liver mitochondria. (1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered. | [
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PMID:7296 | [Photosensitization of consitutents of nucleic acids by proflavine. Mechanism of formation of hydrogen addition radicals in frozen aquenous solutions (author's transl)]. | It is shown that the insertion of nucleotides between proflavine molecules is favourable to photosensitization. Furthermore (1) each molecule of proflavine gives at the most one free radical in the substrate, (2) the chromophore is largely restored when oxygen is not present, (3) superoxide radicals are observed in the presence of oxygen, and (4) formyl radicals are detected. The scheme elaborated for the mechanism gives an explanation for all these observations. | [Photosensitization of consitutents of nucleic acids by proflavine. Mechanism of formation of hydrogen addition radicals in frozen aquenous solutions (author's transl)]. It is shown that the insertion of nucleotides between proflavine molecules is favourable to photosensitization. Furthermore (1) each molecule of proflavine gives at the most one free radical in the substrate, (2) the chromophore is largely restored when oxygen is not present, (3) superoxide radicals are observed in the presence of oxygen, and (4) formyl radicals are detected. The scheme elaborated for the mechanism gives an explanation for all these observations. | [
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PMID:7297 | NADPH/NADP+ ratios in photosynthesizing reconstituted chloroplasts. | Levels of reduced and oxidized triphosphopyridine nucleotides have been determined in reconstituted spinach chloroplasts and compared with levels in whole isolated chloroplasts during photosynthesis and darkness. The ratio of NADPH/NADP+ reaches values slightly above 1.0 at the beginning of photosynthesis, less than half the ratio attained with whole chloroplasts. Nonetheless these lower ratios are sufficient to maintain high rates of photosynthetic carbon dioxide fixation and reduction, which are comparable in the reconstituted chloroplasts to the rates found with whole chloroplasts. As with whole chloroplasts there is a decline in the ration of NADPH/NADP+ as a function of time of photosynthesis. The effect of addition of bicarbonate (6 mM) in causing a transient drop in the ratio of NADPH/NADP/ is described and discussed in terms of the reversibility of the reduction of 3-phosphoglycerate to triose phosphate. The ratio NADPH/NADP+ can be improved by the addition of more lamellae either before or during the course of photosynthesis, and this improvement in ratio is accompanied by an improved rate of CO2 fixation or a more sustained rate of CO2 fixation with time of photosynthesis. The importance of NADPH/NADP+ ratio not only to the reduction of 3-phosphoglycerate to triose phosphate but also to the activation of the ribulose-1,5-diphosphate carboxylasemediated step is discussed. | NADPH/NADP+ ratios in photosynthesizing reconstituted chloroplasts. Levels of reduced and oxidized triphosphopyridine nucleotides have been determined in reconstituted spinach chloroplasts and compared with levels in whole isolated chloroplasts during photosynthesis and darkness. The ratio of NADPH/NADP+ reaches values slightly above 1.0 at the beginning of photosynthesis, less than half the ratio attained with whole chloroplasts. Nonetheless these lower ratios are sufficient to maintain high rates of photosynthetic carbon dioxide fixation and reduction, which are comparable in the reconstituted chloroplasts to the rates found with whole chloroplasts. As with whole chloroplasts there is a decline in the ration of NADPH/NADP+ as a function of time of photosynthesis. The effect of addition of bicarbonate (6 mM) in causing a transient drop in the ratio of NADPH/NADP/ is described and discussed in terms of the reversibility of the reduction of 3-phosphoglycerate to triose phosphate. The ratio NADPH/NADP+ can be improved by the addition of more lamellae either before or during the course of photosynthesis, and this improvement in ratio is accompanied by an improved rate of CO2 fixation or a more sustained rate of CO2 fixation with time of photosynthesis. The importance of NADPH/NADP+ ratio not only to the reduction of 3-phosphoglycerate to triose phosphate but also to the activation of the ribulose-1,5-diphosphate carboxylasemediated step is discussed. | [
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PMID:7298 | Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells. | The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10(-3) M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10(-3) M) only partially reproduced the effect. | Long-term regulation by theophylline of fatty acid synthetase, acetyl-CoA carboxylase and lipid synthesis in cultured glial cells. The long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase and of fatty acid and sterol synthesis was studied in C-6 glial cells in culture. When theophylline (10(-3) M) was added to the culture medium of these cells, rates of lipid synthesis from acetate and activities of synthetase and carboxylase became distinctly lower than in cells that were untreated. This effect appeared after approximately 12 h, and after 48 h enzymatic activities were reduced approx. 2-fold and rates of lipid synthesis from acetate 3- to 4-fold. The likelihood that the decrease in fatty acid synthesis from acetate was caused by the decrease in activities of fatty acid synthetase and acetyl-CoA carboxylase was established by several observations. These indicated that the locus of the effect probably did not reside at the level of acetate uptake into the cell, alterations in acetate pool sizes or conversion of acetate to acetyl-CoA. Moreover, de novo fatty acid synthesis was found to be the predominant pathway in these glial cells, whether treated with theophylline or not. The mechanism of the effect of theophylline on fatty acid synthetase was shown by immunochemical techniques to involve an alteration in content of enzyme rather than in catalytic efficiency. The change in content of fatty acid synthetase was shown by isotopic-immunochemical experiments to involve a decrease in synthesis of the enzyme. The mechanism whereby theophylline leads to a decrease in lipogenesis and in the synthesis of fatty acid synthetase may not be mediated entirely by inhibition of phosphodiesterase and an increase in cyclic AMP levels, because dibutyryl cyclic AMP (10(-3) M) only partially reproduced the effect. | [
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PMID:7299 | Involvement of a single hydroxylase species in the hydroxylation of palmitate at the omega-1, omega-2 and omega-3 positions by a preparation from Bacillus megaterium. | A soluble enzyme preparation from Bacillus megaterium, requiring NADPH and O2 for activity and containing ferredoxin-replaceable and cytochrome P-450-type components, was previously shown to catalyze the conversion of palmitic acid to an isomeric mixture of omega-1, omega-2 and omega-3 hydroxypalmitate. It has now been shown that the ratio of these three positional isomers in the enzymatic product remains unchanged in spite of partial diminution of total hydroxylase activity by heat treatment, pH change or inhibition by p-hydroxy-mercuribenzoate or carbon monoxide. These findings strongly support the hypothesis that a single hydroxylase with one substrate binding site is responsible for hydroxylation at all three positions of palmitate. | Involvement of a single hydroxylase species in the hydroxylation of palmitate at the omega-1, omega-2 and omega-3 positions by a preparation from Bacillus megaterium. A soluble enzyme preparation from Bacillus megaterium, requiring NADPH and O2 for activity and containing ferredoxin-replaceable and cytochrome P-450-type components, was previously shown to catalyze the conversion of palmitic acid to an isomeric mixture of omega-1, omega-2 and omega-3 hydroxypalmitate. It has now been shown that the ratio of these three positional isomers in the enzymatic product remains unchanged in spite of partial diminution of total hydroxylase activity by heat treatment, pH change or inhibition by p-hydroxy-mercuribenzoate or carbon monoxide. These findings strongly support the hypothesis that a single hydroxylase with one substrate binding site is responsible for hydroxylation at all three positions of palmitate. | [
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PMID:7300 | Purification and properties of two lipases from pig adipose tissue. | Two lipases were purified from pig adipose tissue after delipidation by a mild and effective procedure using mixtures of chloroform and butanol. This was followed by hydrophobic adsorption chromatography on aminohexyl-Sepharose 4B coupled with octanoic acid, gel filtration on Sephadex G-100, and isoelectric focusing. Two electrophoretically and chromatographically pure enzymes were obtained, which had the same molecular weight (60 000 +/- 3000) and specific activity, and almost identical amino acid compositions; the isoelectric points, i.e. 5.2 and 5.5, differed. | Purification and properties of two lipases from pig adipose tissue. Two lipases were purified from pig adipose tissue after delipidation by a mild and effective procedure using mixtures of chloroform and butanol. This was followed by hydrophobic adsorption chromatography on aminohexyl-Sepharose 4B coupled with octanoic acid, gel filtration on Sephadex G-100, and isoelectric focusing. Two electrophoretically and chromatographically pure enzymes were obtained, which had the same molecular weight (60 000 +/- 3000) and specific activity, and almost identical amino acid compositions; the isoelectric points, i.e. 5.2 and 5.5, differed. | [
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PMID:7301 | Estrogen biosynthesis and 1beta-hydroxylation using C19 and 19-nor steroid precursors. | (1) In order to study the relationship between aromatization (estrogen biosynthesis) and 1beta-hydroxylation, the effects of a variety of factors on these processes were evaluated. (2) Using the C18 substrate, 4-estrene-3,17-dione, it was found that carbon monoxide, SU-4885, amphenone B, potassium cyanide, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione inhibited the above transformations significantly and to varying degrees. However, within a given experiment the inhibition of each process was similar. (3) SKF-525A did not inhibit either transformation. In addition, phosphate, Tris and barbital buffers, as well as pH changes from 6.9 to 7.7, had no stimulatory or inhibitory effect on the production of estrogen and 1beta-hydroxy compounds. (4) In contrast, several inhibitors affected the aromatization of C19 and C18 steroids differently. These include carbon monoxide, SU-4885 and amphenone B. (5) When a mixture of 4-[7beta-3Hi1estrene-3,17-dione and 19-[4-14C]nortestosterone were incubated together the former was preferentially converted to estrogen. This preference for the 17-keto steroidal form mimics results observed for C19 substrates. (6) We conclude that while estrogen biosynthesis and 1beta-hydroxylation appear to be mediated by the same enzyme system, the same conclusion cannot be drawn for the aromatization of C19 and C18 substrates. | Estrogen biosynthesis and 1beta-hydroxylation using C19 and 19-nor steroid precursors. (1) In order to study the relationship between aromatization (estrogen biosynthesis) and 1beta-hydroxylation, the effects of a variety of factors on these processes were evaluated. (2) Using the C18 substrate, 4-estrene-3,17-dione, it was found that carbon monoxide, SU-4885, amphenone B, potassium cyanide, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione inhibited the above transformations significantly and to varying degrees. However, within a given experiment the inhibition of each process was similar. (3) SKF-525A did not inhibit either transformation. In addition, phosphate, Tris and barbital buffers, as well as pH changes from 6.9 to 7.7, had no stimulatory or inhibitory effect on the production of estrogen and 1beta-hydroxy compounds. (4) In contrast, several inhibitors affected the aromatization of C19 and C18 steroids differently. These include carbon monoxide, SU-4885 and amphenone B. (5) When a mixture of 4-[7beta-3Hi1estrene-3,17-dione and 19-[4-14C]nortestosterone were incubated together the former was preferentially converted to estrogen. This preference for the 17-keto steroidal form mimics results observed for C19 substrates. (6) We conclude that while estrogen biosynthesis and 1beta-hydroxylation appear to be mediated by the same enzyme system, the same conclusion cannot be drawn for the aromatization of C19 and C18 substrates. | [
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PMID:7302 | A sulfonolipid and novel glucosamidyl glycolipids from the extreme thermoacidophile Bacillus acidocaldarius. | The total lipid content of the extreme thermoacidophile Bacillus acidocaldarius comprises about 8.1% of the cell dry weight. Total lipid had a distribution of 15.7% neutral linique component initially characterized as an N-acylglucosamine beta-linked to the primary hydroxyl of an unusual fully saturated pentacyclic triterpene derived tetrol(C35H62O4, Mr 546), which appears to be a derivative of the pentacylcic triterpene hopane substituted at C-29 with a 1,2,3,4-tetrahydroxy pentane. Other major glycolipids present were partially characterized as O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta D-glucopyranosyldiacylglycerol and O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylmonoacylglycerol. Minor components of the glycolipid fraction included O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylglycerol, O-2-amino-2-deoxy-beta-D-glucopyranosyl pentacyclic tetrol and free pentacyclic tetrol. The distributions of esterified and amide-linked fatty acids were similar, being comprised primarily of branched heptadecanoic, 11-cyclohexyundecanoic and 13-cyclohexyltridecanoic acids. The acid lipids were composed of a sulfonoglycosyldiacylglycerol (43.2%), diphosphatidylglycerol (32.3%), lysodiphosphatidylglycerol (5.3%), phosphatidic acid (5.8%) and phosphatidylglycerol (13.4%). | A sulfonolipid and novel glucosamidyl glycolipids from the extreme thermoacidophile Bacillus acidocaldarius. The total lipid content of the extreme thermoacidophile Bacillus acidocaldarius comprises about 8.1% of the cell dry weight. Total lipid had a distribution of 15.7% neutral linique component initially characterized as an N-acylglucosamine beta-linked to the primary hydroxyl of an unusual fully saturated pentacyclic triterpene derived tetrol(C35H62O4, Mr 546), which appears to be a derivative of the pentacylcic triterpene hopane substituted at C-29 with a 1,2,3,4-tetrahydroxy pentane. Other major glycolipids present were partially characterized as O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta D-glucopyranosyldiacylglycerol and O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylmonoacylglycerol. Minor components of the glycolipid fraction included O-beta-D-glucopyranosyl-(1 leads to 4)-O-2-acylamido-2-deoxy-beta-D-glucopyranosylglycerol, O-2-amino-2-deoxy-beta-D-glucopyranosyl pentacyclic tetrol and free pentacyclic tetrol. The distributions of esterified and amide-linked fatty acids were similar, being comprised primarily of branched heptadecanoic, 11-cyclohexyundecanoic and 13-cyclohexyltridecanoic acids. The acid lipids were composed of a sulfonoglycosyldiacylglycerol (43.2%), diphosphatidylglycerol (32.3%), lysodiphosphatidylglycerol (5.3%), phosphatidic acid (5.8%) and phosphatidylglycerol (13.4%). | [
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PMID:7303 | A 1,2,3,4-tetrahydroxy pentane-substituted pentacyclic triterpene from Bacillus acidocaldarius. | A long-chained polyalcohol (polyol) was isolated from the glycolipid fraction of the extreme thermoacidophile Bacillus acidocaldarius. The polyol and its Smith degradation products, as well as the alkanes derived from these compounds were characterized by mobility on thin-layer and gas-liquid chromatography, and by infrared and mass spectrometry. The polyol is proposed to be a fully saturated pentacyclic tetrol (C35H62O4, Mr 546) containing a 1,2,3,4-tetrahydroxy pentane substituted to a hopane-derived pentacyclic triterpene nucleus. | A 1,2,3,4-tetrahydroxy pentane-substituted pentacyclic triterpene from Bacillus acidocaldarius. A long-chained polyalcohol (polyol) was isolated from the glycolipid fraction of the extreme thermoacidophile Bacillus acidocaldarius. The polyol and its Smith degradation products, as well as the alkanes derived from these compounds were characterized by mobility on thin-layer and gas-liquid chromatography, and by infrared and mass spectrometry. The polyol is proposed to be a fully saturated pentacyclic tetrol (C35H62O4, Mr 546) containing a 1,2,3,4-tetrahydroxy pentane substituted to a hopane-derived pentacyclic triterpene nucleus. | [
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PMID:7304 | An investigation of an unusual histidyl residue in Escherichia coli B L-asparaginase through fluorescence quenching. | The tryptophanyl fluorescence of Escherichia coli B L-asparaginase is partially quenched by the protonated form of a base with pKa 6.0 at 25 degrees C, mu = 0.1. This base has been identified as a histidyl residue through the effect of ionic strength and solvent polarity on the pKa. In addition diethylpyrocarbonate which modifies two histidyl residues in the enzyme abolishes the fluorescenc titration and reduces enzymic activity by 90%. The temperature dependence of the histidine pKa is unusual, showing a minimum at 25 degrees C, a thermodynamic analysis of the data shows this to be due to a large negative delta Cp term associated with the ionisation. This is interpreted in terms of the movement of hydrophobic residues into the enzyme on deprotonation of the histidyl residue. The quantum yield of L-asparaginase and its temperature dependence have been measured. The quantum yield is high and there is a low activation energy for radiationless deactivation of the excited state both of which are consistent with a tryptophanyl environment remote from the solvent. | An investigation of an unusual histidyl residue in Escherichia coli B L-asparaginase through fluorescence quenching. The tryptophanyl fluorescence of Escherichia coli B L-asparaginase is partially quenched by the protonated form of a base with pKa 6.0 at 25 degrees C, mu = 0.1. This base has been identified as a histidyl residue through the effect of ionic strength and solvent polarity on the pKa. In addition diethylpyrocarbonate which modifies two histidyl residues in the enzyme abolishes the fluorescenc titration and reduces enzymic activity by 90%. The temperature dependence of the histidine pKa is unusual, showing a minimum at 25 degrees C, a thermodynamic analysis of the data shows this to be due to a large negative delta Cp term associated with the ionisation. This is interpreted in terms of the movement of hydrophobic residues into the enzyme on deprotonation of the histidyl residue. The quantum yield of L-asparaginase and its temperature dependence have been measured. The quantum yield is high and there is a low activation energy for radiationless deactivation of the excited state both of which are consistent with a tryptophanyl environment remote from the solvent. | [
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PMID:7305 | Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius). | Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed. | Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius). Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed. | [
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PMID:7306 | Magnetic circular dichroism of myoglobin-thiolate complexes. | Various complexes of myoglobin (Mb) with thiolate were studied by use of magnetic circular dichroism (MCD) spectroscopy. 1. MetMb-ethyl, n-propyl and isopropylmercaptan complexes offered MCD spectra similar to that of cytochrome P-450 (P-450) with respect to shape and intensity ratio of Soret MCD to Q0-0 MCD. The MCD spectra did not show any pH dependence. The complexes reduced by sodium dithionite exhibited the MCD spectrum of deoxyMb, indicative of release of thiolate anion from the heme iron. 2. Cysteine and cysteine methyl ester coordinated to the heme iron at pH 9.18 but not at pH 6.86 and 11.45. The complex formed at pH 9.18 gave an MCD spectrum similar to that of P-450, and an MCD spectrum of deoxy Mb on reduction with sodium dithionite. 3. The 2-mercaptoethanol complex exhibited three A terms associated with the Q0-0-1, and Soret transitions at pH 6.86 similar to those of Fe(II) cytochrome c, which indicates that Mb was reduced by this reagent at pH 6.86. At pH 9.18 2-mercaptoethanol gave an MCD spectrum similar to that of alkyl mercaptan just after the addition. With the time changed into deoxy Mb through some intermediate of reduced Mb-thiolate complex. At pH 11.45 2-mercaptoethanol formed complex which exhibited an MCD spectrum similar to those of other alkylmercaptans. 4. Sodium sulfide gave an MCD spectrum which resembled that of the normal thiol Mb complex just after addition at pH 6.86. The complex was gradually reduced to give 610 nm trough in addition to the MCD of deoxy Mb. The Mb-sulfur complex formed at pH 9.18 was gradually reduced to give an MCD spectrum which was fairly different from that of deoxy Mb. A similar MCD spectrum was observed at pH 11.45 just after the addition of Na2S. These results were considered to suggest the saturation of one of the conjugated double bonds of the porphyrin by sulfur. | Magnetic circular dichroism of myoglobin-thiolate complexes. Various complexes of myoglobin (Mb) with thiolate were studied by use of magnetic circular dichroism (MCD) spectroscopy. 1. MetMb-ethyl, n-propyl and isopropylmercaptan complexes offered MCD spectra similar to that of cytochrome P-450 (P-450) with respect to shape and intensity ratio of Soret MCD to Q0-0 MCD. The MCD spectra did not show any pH dependence. The complexes reduced by sodium dithionite exhibited the MCD spectrum of deoxyMb, indicative of release of thiolate anion from the heme iron. 2. Cysteine and cysteine methyl ester coordinated to the heme iron at pH 9.18 but not at pH 6.86 and 11.45. The complex formed at pH 9.18 gave an MCD spectrum similar to that of P-450, and an MCD spectrum of deoxy Mb on reduction with sodium dithionite. 3. The 2-mercaptoethanol complex exhibited three A terms associated with the Q0-0-1, and Soret transitions at pH 6.86 similar to those of Fe(II) cytochrome c, which indicates that Mb was reduced by this reagent at pH 6.86. At pH 9.18 2-mercaptoethanol gave an MCD spectrum similar to that of alkyl mercaptan just after the addition. With the time changed into deoxy Mb through some intermediate of reduced Mb-thiolate complex. At pH 11.45 2-mercaptoethanol formed complex which exhibited an MCD spectrum similar to those of other alkylmercaptans. 4. Sodium sulfide gave an MCD spectrum which resembled that of the normal thiol Mb complex just after addition at pH 6.86. The complex was gradually reduced to give 610 nm trough in addition to the MCD of deoxy Mb. The Mb-sulfur complex formed at pH 9.18 was gradually reduced to give an MCD spectrum which was fairly different from that of deoxy Mb. A similar MCD spectrum was observed at pH 11.45 just after the addition of Na2S. These results were considered to suggest the saturation of one of the conjugated double bonds of the porphyrin by sulfur. | [
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PMID:7307 | Structural features of luliberin (luteinising hormone-releasing factor) inferred from fluorescence measurements. | The fluorescence and excitation spectra of luliberin (luteinizing hormone-releasing factor) in 0.005 M aqueous ammonium acetate are identical in shape to those of N-acetyltryptophan amide and are related to the indole side chain of Trp3. The change of fluoresecence intensity of luliberin with pH was measured in the range of pH 4-11. The increase of pH from 4 to 7.5 is followed by about 50% increase in fluorescence intensity due to deprotonation of the imidazolium side chain of His2. The fluorimetric titration curve in this pH region reveals a pK value for His2 of 5.95. Increasing of pH from 8 to 11 results in about 40% quenching of the fluorescence due to electronic energy transfer from the excited indole of Trp3 to the phenolate side chain of Tyr5. The pK value of Tyr5, obtained independently from the fluorimetric and photometric titrations indicate that at pH 7-8 luliberin contains only one charged residue, Arg8, which is in close vicinity to both His2 and Tyr5. The side chains of His2, Tyr5 and Arg8 presumably form a combined unit which may play an active role in the hormone action. Trp3 is at a maximal distance from this unit and may thus act as an independent active unit. | Structural features of luliberin (luteinising hormone-releasing factor) inferred from fluorescence measurements. The fluorescence and excitation spectra of luliberin (luteinizing hormone-releasing factor) in 0.005 M aqueous ammonium acetate are identical in shape to those of N-acetyltryptophan amide and are related to the indole side chain of Trp3. The change of fluoresecence intensity of luliberin with pH was measured in the range of pH 4-11. The increase of pH from 4 to 7.5 is followed by about 50% increase in fluorescence intensity due to deprotonation of the imidazolium side chain of His2. The fluorimetric titration curve in this pH region reveals a pK value for His2 of 5.95. Increasing of pH from 8 to 11 results in about 40% quenching of the fluorescence due to electronic energy transfer from the excited indole of Trp3 to the phenolate side chain of Tyr5. The pK value of Tyr5, obtained independently from the fluorimetric and photometric titrations indicate that at pH 7-8 luliberin contains only one charged residue, Arg8, which is in close vicinity to both His2 and Tyr5. The side chains of His2, Tyr5 and Arg8 presumably form a combined unit which may play an active role in the hormone action. Trp3 is at a maximal distance from this unit and may thus act as an independent active unit. | [
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PMID:7308 | Non-heme iron proteins. The amino acid sequence of rubredoxin from Desulfovibrio vulgaris. | A non-heme iron protein, rubredoxin has been isolated from the sulfate-reducing bacterium, Desulfovibrio vulgaris, strain Hildenborough. The complete amino acid sequence has been established. The 52 amino acid residues of the protein were aligned with the aid of tryptic and chymotryptic peptides and of a fragment produced by cleavage of the Asn-Gly bond (22-23) by hydroxylamine. The sequence of the first 30 residues of the molecule was determined using an automatic sequenator, after removal of the N-terminal methionine by CNBr. In comparing this sequence with those of Micrococcus aerogenes, Clostridium pasteurianum and Peptostreptococcus elsdenii rubredoxins, a high degree of mutation was observed between these homologous proteins. It has been shown that 20 amino acid residues occurred in identical positions. The locations of the four cysteine residues were found to be invariable. A crystallographic study of the Desulfovibrio vulgaris rubredoxin is in progress. | Non-heme iron proteins. The amino acid sequence of rubredoxin from Desulfovibrio vulgaris. A non-heme iron protein, rubredoxin has been isolated from the sulfate-reducing bacterium, Desulfovibrio vulgaris, strain Hildenborough. The complete amino acid sequence has been established. The 52 amino acid residues of the protein were aligned with the aid of tryptic and chymotryptic peptides and of a fragment produced by cleavage of the Asn-Gly bond (22-23) by hydroxylamine. The sequence of the first 30 residues of the molecule was determined using an automatic sequenator, after removal of the N-terminal methionine by CNBr. In comparing this sequence with those of Micrococcus aerogenes, Clostridium pasteurianum and Peptostreptococcus elsdenii rubredoxins, a high degree of mutation was observed between these homologous proteins. It has been shown that 20 amino acid residues occurred in identical positions. The locations of the four cysteine residues were found to be invariable. A crystallographic study of the Desulfovibrio vulgaris rubredoxin is in progress. | [
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PMID:7309 | The dissociation of human deoxyhemoglobin and mixed state hemoglobin into monomer. | The experimental hybridizations between fully deoxygenated human and canine hemoglobins and between half-ligated human hemoglobin and canine cyanomethemoglobin show that new two hybrids in addition to the parent hemoglobins were clearly formed in the mixtures at the high concentration of KI. Thus, human deoxyhemoglobin under the present conditions is in an equilibrium with three species, tetramer in equilibrium dimer in equilibrium monomer. This means that the deoxyhemoglobin is in R-T equilibrium, and shifts considerably toward the R state under the present conditions. On the other hand, the half-ligated hemoglobin in 1.5 M KI becomes much more dissociable than the deoxy T state and appears to be completely transformed into the R state. Nevertheless, the co-operativity, n, is still high (n = 2.0). | The dissociation of human deoxyhemoglobin and mixed state hemoglobin into monomer. The experimental hybridizations between fully deoxygenated human and canine hemoglobins and between half-ligated human hemoglobin and canine cyanomethemoglobin show that new two hybrids in addition to the parent hemoglobins were clearly formed in the mixtures at the high concentration of KI. Thus, human deoxyhemoglobin under the present conditions is in an equilibrium with three species, tetramer in equilibrium dimer in equilibrium monomer. This means that the deoxyhemoglobin is in R-T equilibrium, and shifts considerably toward the R state under the present conditions. On the other hand, the half-ligated hemoglobin in 1.5 M KI becomes much more dissociable than the deoxy T state and appears to be completely transformed into the R state. Nevertheless, the co-operativity, n, is still high (n = 2.0). | [
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PMID:7310 | The interaction of riboflavin with a protein isolated from hen's egg white: a spectrofluorimetric study. | The interaction of riboflavin with a protein isolated from egg white has been studied spectrofluorimetrically at different pH values. In 0.1 M phosphate buffer pH 7.0; 1:1 complex formation occurs with the association constant Ka = 7.7-10(7) M-1. In the presence of 0.033% sodium dodecyl sulphate, the complex dissociated with a rate constant of 4-10(-2) sec-1 at 29 degrees C. The binding was sensitive to pH and to the antibodies produced against the protein. On lowering the pH from 7 to 4 the binding affinity decreased approximately 100-fold and below pH 4, the binding could not be detected at all. These data, together with those obtained by measuring the fluorescence intensities of riboflavin in presence of N-bromosuccinimide oxidized- and disulphide reduced apoprotein, suggest that carboxyl functions, 1-2 tryptophan residues and 2-3 disulphide bridges are essential for binding. The emission spectra of the protein under different conditions upon excitation at 280 and 295 nm were analyzed to calculate the quantum yield (Q) and the efficiency of energy transfer (e) from tyrosine to tryptophan residues. From these data it was concluded that the energy transfer did not occur with equal efficiency under all conditions and that the tryptophan residues responsible for the riboflavin binding are more accessible to N-bromosuccinimide oxidation than others. | The interaction of riboflavin with a protein isolated from hen's egg white: a spectrofluorimetric study. The interaction of riboflavin with a protein isolated from egg white has been studied spectrofluorimetrically at different pH values. In 0.1 M phosphate buffer pH 7.0; 1:1 complex formation occurs with the association constant Ka = 7.7-10(7) M-1. In the presence of 0.033% sodium dodecyl sulphate, the complex dissociated with a rate constant of 4-10(-2) sec-1 at 29 degrees C. The binding was sensitive to pH and to the antibodies produced against the protein. On lowering the pH from 7 to 4 the binding affinity decreased approximately 100-fold and below pH 4, the binding could not be detected at all. These data, together with those obtained by measuring the fluorescence intensities of riboflavin in presence of N-bromosuccinimide oxidized- and disulphide reduced apoprotein, suggest that carboxyl functions, 1-2 tryptophan residues and 2-3 disulphide bridges are essential for binding. The emission spectra of the protein under different conditions upon excitation at 280 and 295 nm were analyzed to calculate the quantum yield (Q) and the efficiency of energy transfer (e) from tyrosine to tryptophan residues. From these data it was concluded that the energy transfer did not occur with equal efficiency under all conditions and that the tryptophan residues responsible for the riboflavin binding are more accessible to N-bromosuccinimide oxidation than others. | [
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PMID:7311 | Histamine (H2) receptor-adenylate cyclase system in pig skin (epidermis). | Histamine activated adenylate cyclase in pig skin (epidermal) slices, resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of cyclic AMP-phosphodiesterase inhibitors (theophylline, papaverine). A specific H2 receptor inhibitor (metiamide) inhibited the effect of histamine completely, while other antihistamines (diphenhydramine, acetophenazine, perphenazine, fluphenazine, promethazine) inhibited the effect of histamine to various lesser degrees. It has been shown that both epinephrine and prostaglandin E stimulate epidermal adenylate cyclase. Our data using specific blocking agents indicate that histamine, epinephrine and prostaglandin E2 act independently on the epidermal adenylate cyclase system. | Histamine (H2) receptor-adenylate cyclase system in pig skin (epidermis). Histamine activated adenylate cyclase in pig skin (epidermal) slices, resulting in the accumulation of cyclic AMP. This effect was highly potentiated by the addition of cyclic AMP-phosphodiesterase inhibitors (theophylline, papaverine). A specific H2 receptor inhibitor (metiamide) inhibited the effect of histamine completely, while other antihistamines (diphenhydramine, acetophenazine, perphenazine, fluphenazine, promethazine) inhibited the effect of histamine to various lesser degrees. It has been shown that both epinephrine and prostaglandin E stimulate epidermal adenylate cyclase. Our data using specific blocking agents indicate that histamine, epinephrine and prostaglandin E2 act independently on the epidermal adenylate cyclase system. | [
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PMID:7312 | Ammonia production by the small intestine of the rat. | 1. Slices of duodenum and jejunum produce ammonia from glutamine in vitro. 2. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. 3. Gut contains glutaminase 1 as well as gamma-glutamyl transpeptidase. 4. These enzymes do not show any increase during starvation. | Ammonia production by the small intestine of the rat. 1. Slices of duodenum and jejunum produce ammonia from glutamine in vitro. 2. Ammoniagenesis does not increase in response to acidosis or potassium deficiency, two conditions known to cause enhanced ammoniagenesis in the kidney. 3. Gut contains glutaminase 1 as well as gamma-glutamyl transpeptidase. 4. These enzymes do not show any increase during starvation. | [
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PMID:7313 | pH dependence of 13C-15N coupling constants of highly 14N-enriched amino acids isolated from mass cultivation of algae. | The alga Ankistrodesmus braunii was grown with [14N]nitrate under optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The 15N-labelled amino acids (15N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The 15N cotent of the analytically pure amino acid was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the 15N analysator. Using pulse Fourier transform 13C nuclear magnetic resonance, the pH dependence of the 13C-15N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J.Am. Chem. Soc. 86,5564-5570) between the coupling constant and the product of the S-part of the 13C and 15N hybridization SC - SN = 80 - J (13C-45X) fits best in acidic medium. The magnitude of coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153-182). The recording of 13C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the 13C-15N coupling constants of the amino acids. | pH dependence of 13C-15N coupling constants of highly 14N-enriched amino acids isolated from mass cultivation of algae. The alga Ankistrodesmus braunii was grown with [14N]nitrate under optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The 15N-labelled amino acids (15N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The 15N cotent of the analytically pure amino acid was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the 15N analysator. Using pulse Fourier transform 13C nuclear magnetic resonance, the pH dependence of the 13C-15N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J.Am. Chem. Soc. 86,5564-5570) between the coupling constant and the product of the S-part of the 13C and 15N hybridization SC - SN = 80 - J (13C-45X) fits best in acidic medium. The magnitude of coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153-182). The recording of 13C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the 13C-15N coupling constants of the amino acids. | [
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PMID:7314 | Secondary kinase reactions catalyzed by yeast pyruvate kinase. | 1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed. | Secondary kinase reactions catalyzed by yeast pyruvate kinase. 1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed. | [
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PMID:7315 | The proton transfer reactions catalyzed by yeast pyruvate kinase. | 1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton-transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10-100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction of pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase (Robinson, JL. and Rose, I.A. (1972) J. Biol. Chem. 247, 1096-1105). The pH-profiles and the divalent cation dependencies were similar for Fru-1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization by yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization by the muscle enzyme which proceeds when ATP is replaced by Pi or other dianions. 3. Fructose-1,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme. | The proton transfer reactions catalyzed by yeast pyruvate kinase. 1. The proton-transfer reactions of yeast pyruvate kinase (EC 2.7.1.40) were studied. Proton-transfer from C-3 of phosphoenolpyruvate to water occurs only in the presence of the phosphoryl-acceptor ADP. Proton transfer from C-3 of pyruvate to water occurs only in the presence of ATP. However, the proton transfer in the latter case occurs 10-100 times faster than phosphoryl transfer; this supports a mechanism in which proton transfer precedes phosphoryl transfer in the reverse reaction of pyruvate kinase. 2. The characteristics of proton-transfer reactions of yeast pyruvate kinase were compared with those previously reported for rabbit muscle pyruvate kinase (Robinson, JL. and Rose, I.A. (1972) J. Biol. Chem. 247, 1096-1105). The pH-profiles and the divalent cation dependencies were similar for Fru-1,6-P2-activated yeast pyruvate kinase and the muscle enzyme. Pyruvate enolization by yeast pyruvate kinase has an absolute requirement for ATP in contrast to enolization by the muscle enzyme which proceeds when ATP is replaced by Pi or other dianions. 3. Fructose-1,6-bisphosphate was shown to affect the catelytic steps of yeast pyruvate kinase in addition to the binding of substrates. Its role depends on the divalent cation used to activate the enzyme. | [
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PMID:7316 | Importance of SH groups in catalysis by bovine brain acid phosphatase. | The rate of inactivation of acid phosphatase (EC 3.1.3.2) from bovine brain by dithiobis-(2-nitrobenzoic acid) (Nbs2) is identical to the rate of titration of one of the two SH groups of this enzyme. The rate of inactivation of the enzyme by Nbs2 is pH dependent and, at 300 mM NaCl, can be described by the reaction of a single SH group of pK 8.4. At low ionic strength the pK determined from the k inactivation vs. pH profile is 7.7 and the results deviate markedly from the predicted values at pH values less than or equal to 6. The decrease of V upon addition of salts is paralleled by the decrease of inactivation rate by Nbs2. The relevance of SH groups in catalysis by bovine brain acid phosphatase is discussed in terms of these data. | Importance of SH groups in catalysis by bovine brain acid phosphatase. The rate of inactivation of acid phosphatase (EC 3.1.3.2) from bovine brain by dithiobis-(2-nitrobenzoic acid) (Nbs2) is identical to the rate of titration of one of the two SH groups of this enzyme. The rate of inactivation of the enzyme by Nbs2 is pH dependent and, at 300 mM NaCl, can be described by the reaction of a single SH group of pK 8.4. At low ionic strength the pK determined from the k inactivation vs. pH profile is 7.7 and the results deviate markedly from the predicted values at pH values less than or equal to 6. The decrease of V upon addition of salts is paralleled by the decrease of inactivation rate by Nbs2. The relevance of SH groups in catalysis by bovine brain acid phosphatase is discussed in terms of these data. | [
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PMID:7317 | Guanylate cyclase. Existence of different forms and their regulation by nucleotides in calf uterus. | The activity of calf uterus guanylate cyclase (EC 4.6.1.2) exists in at least two and most probably three distinct forms. The cytosolic enzyme exhibits hyperbolic substrate curves with respect to GTP and Mn2+, while the particulate cyclases (nuclear and microsomal)display sigmoidal (GTP) and hyperbolic (Mn2+) relationships. The Hill coefficient for the GTP dependence is 0.9 for the cytosolic, 1.5 for the nuclear, and 1.4 for the microsomal enzyme. The cytosolic enzyme has a Km for GTP of 70 muM while half maximal velocity occurs at 90 and 100 muM GTP for the nuclear and microsomal enzymes, respectively. The Ka for Mn2+ is 0.57, 0.71 or 0.75 mM for the cytosolic, nuclear, or microsomal enzyme, respectively. | Guanylate cyclase. Existence of different forms and their regulation by nucleotides in calf uterus. The activity of calf uterus guanylate cyclase (EC 4.6.1.2) exists in at least two and most probably three distinct forms. The cytosolic enzyme exhibits hyperbolic substrate curves with respect to GTP and Mn2+, while the particulate cyclases (nuclear and microsomal)display sigmoidal (GTP) and hyperbolic (Mn2+) relationships. The Hill coefficient for the GTP dependence is 0.9 for the cytosolic, 1.5 for the nuclear, and 1.4 for the microsomal enzyme. The cytosolic enzyme has a Km for GTP of 70 muM while half maximal velocity occurs at 90 and 100 muM GTP for the nuclear and microsomal enzymes, respectively. The Ka for Mn2+ is 0.57, 0.71 or 0.75 mM for the cytosolic, nuclear, or microsomal enzyme, respectively. | [
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PMID:7318 | Properties of highly purified lysyl oxidase from embryonic chick cartilage. | Lysyl oxidase the enzyme which oxidately deaminates lysine residues in collagen and elastin, was purified from embryonic chick cartialge by employing an affinity column of lathyritic rat skin collagen coupled to Sepharose, followed by separation on DEAE-cellulose. An enzyme preparation was obtained which was pure as shown by polyacrylamide gel electrophoresis. The specific activity was 1800-fold higher than that of the original extract. The pure enzyme utilized both collagen and elastin substrate. Furthermore, the ratios of enzyme activity with elastin substrate versus that with collagen substrate were the same at all stages of purity. Only one protein band was found after polyacrylamide gel electrophoresis of the pure lysyl oxidase in sodium dodecyl sulfate and mercaptoethanol. The molecular weight was estimated to be 28000. It was found that the enzyme contained a large number of cysteine and tyrosine residues. Evidence was obtained for molecular heterogeneity of lysyl oxidase. The enzyme eluted from DEAE-cellulsoe in at least four distinct regions. When the peaks were rechromatographed separately, they eluted at salt concentrations similar to those of the original chromatogram. However, the substrate specificity and the electrophoretic mobility on polyacrylamide gel were the same for all enzyme fractions. | Properties of highly purified lysyl oxidase from embryonic chick cartilage. Lysyl oxidase the enzyme which oxidately deaminates lysine residues in collagen and elastin, was purified from embryonic chick cartialge by employing an affinity column of lathyritic rat skin collagen coupled to Sepharose, followed by separation on DEAE-cellulose. An enzyme preparation was obtained which was pure as shown by polyacrylamide gel electrophoresis. The specific activity was 1800-fold higher than that of the original extract. The pure enzyme utilized both collagen and elastin substrate. Furthermore, the ratios of enzyme activity with elastin substrate versus that with collagen substrate were the same at all stages of purity. Only one protein band was found after polyacrylamide gel electrophoresis of the pure lysyl oxidase in sodium dodecyl sulfate and mercaptoethanol. The molecular weight was estimated to be 28000. It was found that the enzyme contained a large number of cysteine and tyrosine residues. Evidence was obtained for molecular heterogeneity of lysyl oxidase. The enzyme eluted from DEAE-cellulsoe in at least four distinct regions. When the peaks were rechromatographed separately, they eluted at salt concentrations similar to those of the original chromatogram. However, the substrate specificity and the electrophoretic mobility on polyacrylamide gel were the same for all enzyme fractions. | [
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PMID:7319 | Oxidation of sarcosine and N-alkyl derivatives of glycine by D-amino-acid oxidase. | 1. Sarcosine was oxidized by D-amino-acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) to yield methylamine and glyoxylic acid. A seriies of N-alkyl glycines were also oxidized by this enzyme. 2. N-Acetyl- and N-Phenylglycine inhibited the oxidase by competing with the substrate, while N-methyl-N-acetylglycine did not bind to the enzyme. This suggests the requirement of at least one unsubstituted hydrogen atom at the amino group ofglycine for binding. 3. The primary step in the reaction was the release of a proton from the substrate, indicating the formation of a substituted imino acid, which was spontaneously hydrolyzed to glyoxylic acid acid and an amine. | Oxidation of sarcosine and N-alkyl derivatives of glycine by D-amino-acid oxidase. 1. Sarcosine was oxidized by D-amino-acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) to yield methylamine and glyoxylic acid. A seriies of N-alkyl glycines were also oxidized by this enzyme. 2. N-Acetyl- and N-Phenylglycine inhibited the oxidase by competing with the substrate, while N-methyl-N-acetylglycine did not bind to the enzyme. This suggests the requirement of at least one unsubstituted hydrogen atom at the amino group ofglycine for binding. 3. The primary step in the reaction was the release of a proton from the substrate, indicating the formation of a substituted imino acid, which was spontaneously hydrolyzed to glyoxylic acid acid and an amine. | [
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PMID:7320 | Factors affecting the ADP/O ratio in isolated chloroplasts. | (1) The effect of gradual disruption of the outer membrane of intact chloroplasts on CO2 fixation, electron transport and phosphorylation was investigated. The results suggested that whilst ferricyanide and substrate amounts of ADP enter intact chloroplasts only very slowly, methyl viologen rapidly penetrates the outer membrane. (2) Preparatwons of intact pea chloroplasts had an ATP-consuming reaction which resulted in decreased ADP/O ratios when noncyclic electron transport was measured after disruption of the outer membrane. The ATP-consuming reaction was removed into the supernatant after washing the disrupted chloroplasts. The resulting washed chloroplasts gave ADP/O ratios of 1.5-1.6 for ferricyanide and 1.9-2.0 for methyl viologen. (3) Preparations of intact spinach chloroplasts had lower activity of the ATP-consuming reaction and gave similar ADP/O ratios to washed pea chloroplasts. The ADP/O ratios of spinach chloroplasts did not alter significantly after washing. (4) An investigation of the effect of various assay conditions on the ADP/O ratio showed that the phosphate concentration was critical in obtaining optimal values for ADP/O ratio. Decreasing the phosphate concentration below 10 mM decreased the ADP/O ratio significantly. (5) It is suggested that the maximum ADP/O ratio of chloroplasts is 2.0 but that lower values can be obtained in the presence of an ATP-consuming reaction, under suboptimal assay conditions or where the chloroplasts are structurally damaged. | Factors affecting the ADP/O ratio in isolated chloroplasts. (1) The effect of gradual disruption of the outer membrane of intact chloroplasts on CO2 fixation, electron transport and phosphorylation was investigated. The results suggested that whilst ferricyanide and substrate amounts of ADP enter intact chloroplasts only very slowly, methyl viologen rapidly penetrates the outer membrane. (2) Preparatwons of intact pea chloroplasts had an ATP-consuming reaction which resulted in decreased ADP/O ratios when noncyclic electron transport was measured after disruption of the outer membrane. The ATP-consuming reaction was removed into the supernatant after washing the disrupted chloroplasts. The resulting washed chloroplasts gave ADP/O ratios of 1.5-1.6 for ferricyanide and 1.9-2.0 for methyl viologen. (3) Preparations of intact spinach chloroplasts had lower activity of the ATP-consuming reaction and gave similar ADP/O ratios to washed pea chloroplasts. The ADP/O ratios of spinach chloroplasts did not alter significantly after washing. (4) An investigation of the effect of various assay conditions on the ADP/O ratio showed that the phosphate concentration was critical in obtaining optimal values for ADP/O ratio. Decreasing the phosphate concentration below 10 mM decreased the ADP/O ratio significantly. (5) It is suggested that the maximum ADP/O ratio of chloroplasts is 2.0 but that lower values can be obtained in the presence of an ATP-consuming reaction, under suboptimal assay conditions or where the chloroplasts are structurally damaged. | [
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PMID:7321 | Photosynthesis in a reconstituted chloroplast system from spinach. Some factors affecting CO2-dependent oxygen evolution with fructose-1,6-bisphosphate as substrate. | When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, 14CO2 fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O2 evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway. | Photosynthesis in a reconstituted chloroplast system from spinach. Some factors affecting CO2-dependent oxygen evolution with fructose-1,6-bisphosphate as substrate. When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, 14CO2 fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O2 evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway. | [
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PMID:7322 | Light-driven proton translocations in Halobacterium halobium. | The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses. | Light-driven proton translocations in Halobacterium halobium. The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses. | [
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PMID:7323 | Primary reactions of photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts. | The effects of lowering the pH on Photosystem II has been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts. At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese. The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H.J., Pulles, M.P.J., Haveman, J. and den Haan, G.A. (1976) Biochim. Biophys, Acta 423, 217-226). | Primary reactions of photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts. The effects of lowering the pH on Photosystem II has been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts. At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese. The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H.J., Pulles, M.P.J., Haveman, J. and den Haan, G.A. (1976) Biochim. Biophys, Acta 423, 217-226). | [
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PMID:7324 | Ca++ binding properties of human prothrombin. | The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin. | Ca++ binding properties of human prothrombin. The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin. | [
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PMID:7325 | Structure-volume relationships in hemoglobin. A densitometric and dilatometric study of the oxy leads to deoxy transformation. | Partial volume measurements have been performed for human hemoglobin, both on the oxygenated and deoxygenated forms. Density measurements (by pycnometry) give vHbO2 = 0.752 +/- 0.002 and vHb =0.753 +/- 0.006 for the partial specific volume and do not distinguish between the two different structures. Differential measurements, by dilatometry, however, show a signigicantly higher molal volume (of about 50 cm3/mol hemoglobin tetramer) for the deoxy over the oxygenated from at pH 7. The same reaction, at pH 9, gives a much smaller increase or even a decrease of volume. The different volume changes at pH 7 and at pH 9 are not due to the so-called Bohr ionization but to the weakening, at pH 9 compared to pH 7, of stabilising salt linkages in the deoxy structure. | Structure-volume relationships in hemoglobin. A densitometric and dilatometric study of the oxy leads to deoxy transformation. Partial volume measurements have been performed for human hemoglobin, both on the oxygenated and deoxygenated forms. Density measurements (by pycnometry) give vHbO2 = 0.752 +/- 0.002 and vHb =0.753 +/- 0.006 for the partial specific volume and do not distinguish between the two different structures. Differential measurements, by dilatometry, however, show a signigicantly higher molal volume (of about 50 cm3/mol hemoglobin tetramer) for the deoxy over the oxygenated from at pH 7. The same reaction, at pH 9, gives a much smaller increase or even a decrease of volume. The different volume changes at pH 7 and at pH 9 are not due to the so-called Bohr ionization but to the weakening, at pH 9 compared to pH 7, of stabilising salt linkages in the deoxy structure. | [
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PMID:7326 | Thermodynamics of hexokinase catalyzed reactions. II. Measurement and calculation of enthalpies of reaction as a function of magnesium ion concentration. | Enthalpies of phosphorylation of glucose by adenosine 5'-triphosphate have been measured as a function of concentrations of magnesium chloride in TRIS/TRIS-HCl buffer in the pH range 8.64 to 8.98. These measurements are compared with the results of calculations of these enthalpies that use a coupled equilibrium formalism with equilibrium data and enthalpy values selected from the literature. The experimental results span the range of magnesium ion concentrations 1 X 10(-6) to 0.3 mol alpha-1 and show a total variation in the enthalpy of reaction of almost 10 kJ mol-1, with the most exothermic reaction occurring at a magnesium ion concentration of 6.0 X 10(-4) mol alpha-1. The calculated enthalpies of reaction, except for the magnesium ion concentration range 4 X 10(-6) to 5 X 10(-4) mol alpha-1, are, within estimated uncertainty intervals (0.8 to 10.2 kJ mol-1), in agreement with the measured values. | Thermodynamics of hexokinase catalyzed reactions. II. Measurement and calculation of enthalpies of reaction as a function of magnesium ion concentration. Enthalpies of phosphorylation of glucose by adenosine 5'-triphosphate have been measured as a function of concentrations of magnesium chloride in TRIS/TRIS-HCl buffer in the pH range 8.64 to 8.98. These measurements are compared with the results of calculations of these enthalpies that use a coupled equilibrium formalism with equilibrium data and enthalpy values selected from the literature. The experimental results span the range of magnesium ion concentrations 1 X 10(-6) to 0.3 mol alpha-1 and show a total variation in the enthalpy of reaction of almost 10 kJ mol-1, with the most exothermic reaction occurring at a magnesium ion concentration of 6.0 X 10(-4) mol alpha-1. The calculated enthalpies of reaction, except for the magnesium ion concentration range 4 X 10(-6) to 5 X 10(-4) mol alpha-1, are, within estimated uncertainty intervals (0.8 to 10.2 kJ mol-1), in agreement with the measured values. | [
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PMID:7327 | The influence of organic phosphates on the Bohr effect of human hemoglobin valency hybrids. | The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaphosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the alpha chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized beta chains is slighly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species. | The influence of organic phosphates on the Bohr effect of human hemoglobin valency hybrids. The Bohr effect of hemoglobin and that of the aquomet and cyanomet valency hybrids was measured in the presence and the absence of IHP (inositol hexaphosphate) and DPG (2,3-diphosphoglycerate). In the absence of these organic phosphates the four hybrids show similar, but suppressed Bohr effects as compared to hemoglobin. Addition of IHP and DPG results in all cases in an increase of the Bohr effect. The additional phosphate induced Bohr effect of the hybrids with the alpha chain in the oxidized form is almost identical to that of hemoglobin, while this effect of the hybrids with oxidized beta chains is slighly lower than that of hemoglobin. The results suggest (a) that the Bohr effect is correlated to the ligation state of the hemoglobin molecule rather than to its quaternary structure (b) that the additional phosphate induced Bohr effect is related to the change in quaternary structure of the tetramer, and (c) that with respect to the Bohr effect of the hybrids there is no difference between high and low spin species. | [
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PMID:7328 | [Effect of blocking the neuronal and extraneuronal uptake of bioamines on the adrenosensitizing action of tricyclic antidepressants]. | Tests conducted on isolated and denervated preparations of the rat seminal duct brought evidence that tricyclic antidepressants (melipromine, noverile and azaphen) when employed in low concentrations (1-10(-9) g/ml) produced an adrenosensitizing effect. Denervation with the subsequent block by desoxycorticosterone (1-10(-5) g/ml) of exteraneuronal amine uptake did not alter the position, shape and inclination of the "concentration-effect" noradrenaline curves received in the presence of noverile and cocaine. It is believed that there exists a predominance of the postsynaptic mechanism of the aminosensitizing action of tricyclic antidepressants on the smooth muscle organ. | [Effect of blocking the neuronal and extraneuronal uptake of bioamines on the adrenosensitizing action of tricyclic antidepressants]. Tests conducted on isolated and denervated preparations of the rat seminal duct brought evidence that tricyclic antidepressants (melipromine, noverile and azaphen) when employed in low concentrations (1-10(-9) g/ml) produced an adrenosensitizing effect. Denervation with the subsequent block by desoxycorticosterone (1-10(-5) g/ml) of exteraneuronal amine uptake did not alter the position, shape and inclination of the "concentration-effect" noradrenaline curves received in the presence of noverile and cocaine. It is believed that there exists a predominance of the postsynaptic mechanism of the aminosensitizing action of tricyclic antidepressants on the smooth muscle organ. | [
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PMID:7329 | [Capability of lymphocytes, stimulated in vitro with phytohemagglutinins to accomplish the graft versus host reaction]. | Lymph node cells from CBA mice cultivated in the presence of PHA for 2 hours proved to be more potent, and for 44 hours--less potent as compared with normal non-cultivated cells in their capacity to realize the GVHR after injection to sublethally irradiated (CBAXC57BL) F1 recipients. Syngeneic or killed allogeneic lymphocytes cultivated similarly and phytohemagglutinin were found to be deprived of this potency. | [Capability of lymphocytes, stimulated in vitro with phytohemagglutinins to accomplish the graft versus host reaction]. Lymph node cells from CBA mice cultivated in the presence of PHA for 2 hours proved to be more potent, and for 44 hours--less potent as compared with normal non-cultivated cells in their capacity to realize the GVHR after injection to sublethally irradiated (CBAXC57BL) F1 recipients. Syngeneic or killed allogeneic lymphocytes cultivated similarly and phytohemagglutinin were found to be deprived of this potency. | [
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PMID:7334 | Selectivity of beta-adrenoceptor agonists and antagonists on bronchial, skeletal, vascular and cardiac muscle in the anaesthetized cat. | 1 The potencies of fifteen beta-adrenoceptor agonists of widely differing chemical structures were compared with that of (-)-isoprenaline on bronchial muscle, soleus muscle, blood pressure and heart rate in the anaesthetized cat. The beta-adrenoceptor antagonist potencies of propranolol and practolol were determined against (-)-isoprenaline in the same model. 2 (-)-Isoprenaline was the most potent agonist and its action was essentially unselective. Thus, on all four parameters the minimal effective dose was 0.003-0.01 mug/kg and maximal or near maximal responses were produced by 0.3-1 mug/kg. Trimetoquinol was also an essentially unselective agonist. 3 For thirteen of the remaining fourteen agonists, potency was similar on bronchial muscle, soleus muscle and blood pressure but significantly lower on heart rate. 4 The remaining agonist - AH 7616 (4-hydroxy-alpha1-[[(1-methyl-3,3-diphenyl-propyl)amino]-methyl]-m-xylene-alpha1, alpha3-diol, acetate) - was also significantly less potent on heart rate than on the other parameters; in addition, it was clearly less potent on soleus muscle and blood pressure than on bronchial muscle when 5-hydroxytryptamine (5-HT) was used to induce bronchospasm. However, when acetylcholine was used instead of 5-HT the potency of AH 7616 on induce bronchospasm. However, when acetylcholine was used instead of 5-HT the potency of AH 7616 on bronchial muscle, soleus muscle and blood pressure was very similar. AH 7616 may therefore possess a specific 5-HT antagonist action in addition to its beta-adrenoceptor agonist action. 5 The fifteen test agonists were longer acting than (-)-isoprenaline and this was particularly true of trimetoquinol and soterenol. 6 The beta-adrenoceptor antagonist potency of propranolol was almost identical on bronchial muscle, soleus muscle and blood pressure and very slightly lower on the heart. Practolol was 10-12 times more potent on the heart than on bronchial muscle, soleus muscle and blood pressure. 7 These findings suggest that it may not be possible to separate the bronchodilating and tremorenhancing properties of beta-adrenoceptor agonists. The results with agonists and antagonists are in accord with Lands' dual beta-adrenoceptor sub-classification. | Selectivity of beta-adrenoceptor agonists and antagonists on bronchial, skeletal, vascular and cardiac muscle in the anaesthetized cat. 1 The potencies of fifteen beta-adrenoceptor agonists of widely differing chemical structures were compared with that of (-)-isoprenaline on bronchial muscle, soleus muscle, blood pressure and heart rate in the anaesthetized cat. The beta-adrenoceptor antagonist potencies of propranolol and practolol were determined against (-)-isoprenaline in the same model. 2 (-)-Isoprenaline was the most potent agonist and its action was essentially unselective. Thus, on all four parameters the minimal effective dose was 0.003-0.01 mug/kg and maximal or near maximal responses were produced by 0.3-1 mug/kg. Trimetoquinol was also an essentially unselective agonist. 3 For thirteen of the remaining fourteen agonists, potency was similar on bronchial muscle, soleus muscle and blood pressure but significantly lower on heart rate. 4 The remaining agonist - AH 7616 (4-hydroxy-alpha1-[[(1-methyl-3,3-diphenyl-propyl)amino]-methyl]-m-xylene-alpha1, alpha3-diol, acetate) - was also significantly less potent on heart rate than on the other parameters; in addition, it was clearly less potent on soleus muscle and blood pressure than on bronchial muscle when 5-hydroxytryptamine (5-HT) was used to induce bronchospasm. However, when acetylcholine was used instead of 5-HT the potency of AH 7616 on induce bronchospasm. However, when acetylcholine was used instead of 5-HT the potency of AH 7616 on bronchial muscle, soleus muscle and blood pressure was very similar. AH 7616 may therefore possess a specific 5-HT antagonist action in addition to its beta-adrenoceptor agonist action. 5 The fifteen test agonists were longer acting than (-)-isoprenaline and this was particularly true of trimetoquinol and soterenol. 6 The beta-adrenoceptor antagonist potency of propranolol was almost identical on bronchial muscle, soleus muscle and blood pressure and very slightly lower on the heart. Practolol was 10-12 times more potent on the heart than on bronchial muscle, soleus muscle and blood pressure. 7 These findings suggest that it may not be possible to separate the bronchodilating and tremorenhancing properties of beta-adrenoceptor agonists. The results with agonists and antagonists are in accord with Lands' dual beta-adrenoceptor sub-classification. | [
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PMID:7332 | A method of continuous recording on microsamples of the Hb-O2 association curve. I. Technique and direct registration of standard results. | A modified technique for continuous recording of the Hb-O2 association curve for blood samples is proposed. The following alterations to the classical method of Duvelleroy et al. (5) were studied and validated: 1) dilution of blood micro-samples (200-400 mul) to 10 ml of a buffered phosphate solution; 2) use of a (CO2, O2) mixture allowing the maintenance of standard conditions (pH=7.40, PCO2=40 Torr) during the entire oxygenation process. The advantages of such modifications were: 1) reducing the time necessary for both deoxygenation and drawing Hb-O2 association curve (to about 20 min), hence permitting to work out large series of samples; 2) avoiding the necessity of imprecise a posteriori corrections, thus permitting analysis of the different components of the BOHR effect. | A method of continuous recording on microsamples of the Hb-O2 association curve. I. Technique and direct registration of standard results. A modified technique for continuous recording of the Hb-O2 association curve for blood samples is proposed. The following alterations to the classical method of Duvelleroy et al. (5) were studied and validated: 1) dilution of blood micro-samples (200-400 mul) to 10 ml of a buffered phosphate solution; 2) use of a (CO2, O2) mixture allowing the maintenance of standard conditions (pH=7.40, PCO2=40 Torr) during the entire oxygenation process. The advantages of such modifications were: 1) reducing the time necessary for both deoxygenation and drawing Hb-O2 association curve (to about 20 min), hence permitting to work out large series of samples; 2) avoiding the necessity of imprecise a posteriori corrections, thus permitting analysis of the different components of the BOHR effect. | [
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PMID:7335 | Blockade by burimamide of the restorative effect of histamine in tetrodotoxin-treated heart preparations. | 1 In isolated heart preparations in which fast sodium channels were blocked by tetrodotoxin (2-4 x 10(-5) M), excitability was restored by histamine (6 x 10(-6) M to 10(-5) M). 2 This effect was antagonized by EDTA (2 X 10(-6) M),D600 compound (0.5mug/ml) and the H2-receptor antagonist, burimamide(2 X 10(-4) M). | Blockade by burimamide of the restorative effect of histamine in tetrodotoxin-treated heart preparations. 1 In isolated heart preparations in which fast sodium channels were blocked by tetrodotoxin (2-4 x 10(-5) M), excitability was restored by histamine (6 x 10(-6) M to 10(-5) M). 2 This effect was antagonized by EDTA (2 X 10(-6) M),D600 compound (0.5mug/ml) and the H2-receptor antagonist, burimamide(2 X 10(-4) M). | [
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PMID:7336 | Investigations to characterize a new anti-arrhythmic drug, ORG 6001 including a simple test for calcium antagonism. | 1 The compound Org 6001 (3alpha-amino-2beta-hydroxy-5alpha-androstan-17-one hydrochloride) was found in recent experiments to exhibit anti-arrhythmic activity. Evidence is presented in this paper concerning its mode of action. 2 Org 6001 was 1.8 times more potent than procaine as a local anaesthetic on desheathed frog nerve. 3 Org 6001 had no effect on the resting potential of isolated cardiac muscle of rabbit, but greatly reduced the maximum rate of depolarization tion (MRD). The action potential duration TAPD) WAS MARGINALLY PROLONGED IN ATRIAL AND VENTRICULAR MUSCLE. 4 Org 6001 preferentially shortened APD in that part of the Purkinje system in which APD is normally longer than elsewhere, so that APD | Investigations to characterize a new anti-arrhythmic drug, ORG 6001 including a simple test for calcium antagonism. 1 The compound Org 6001 (3alpha-amino-2beta-hydroxy-5alpha-androstan-17-one hydrochloride) was found in recent experiments to exhibit anti-arrhythmic activity. Evidence is presented in this paper concerning its mode of action. 2 Org 6001 was 1.8 times more potent than procaine as a local anaesthetic on desheathed frog nerve. 3 Org 6001 had no effect on the resting potential of isolated cardiac muscle of rabbit, but greatly reduced the maximum rate of depolarization tion (MRD). The action potential duration TAPD) WAS MARGINALLY PROLONGED IN ATRIAL AND VENTRICULAR MUSCLE. 4 Org 6001 preferentially shortened APD in that part of the Purkinje system in which APD is normally longer than elsewhere, so that APD | [
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PMID:7333 | [A method of continuous recording on microsamples of the Hb-O2 association curve. II. A study of Bohr effect and carbamino-formation (author's transl)]. | The authors have worked out an adaptation to microsamples (200-400 mul) of the DUVELLEROY et al. method, allowing the continous registration of the O2-Hb association curve. The microsample being diluted in buffer solution, it is possible to predetermine its pH and PCO2. The prefixed conditions are maintained during the initial deoxygenation phase, and all along the curve registration. Moreover, the adjustment to the desired values of the pH and PCO2 allows the quantification of total BOHR effect, proton BOHR effect and carbamino-formation, during the course of oxygenation. | [A method of continuous recording on microsamples of the Hb-O2 association curve. II. A study of Bohr effect and carbamino-formation (author's transl)]. The authors have worked out an adaptation to microsamples (200-400 mul) of the DUVELLEROY et al. method, allowing the continous registration of the O2-Hb association curve. The microsample being diluted in buffer solution, it is possible to predetermine its pH and PCO2. The prefixed conditions are maintained during the initial deoxygenation phase, and all along the curve registration. Moreover, the adjustment to the desired values of the pH and PCO2 allows the quantification of total BOHR effect, proton BOHR effect and carbamino-formation, during the course of oxygenation. | [
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PMID:7337 | The effects of pentobarbitone and pethidine on foetal breathing movements in sheep. | 1 Small doses of pentobarbitone (4 mg/kg i.v.) administered to sheep in the last third of pregancy had little overt effect on the mothers. In the foetus they caused arrest of breathing movements, an alteration in the character of the electrocorticogram and cardiovascular changes which varied with gestational age. 2 In contrast, relatively large doses of pethidine (100-200 mg) admininstered to the mother had no consistent effect on normal foetal breathing movements, though they abolished the foetal response to hypercapnia. 3 The results are discussed in relation to feotal sleep state. | The effects of pentobarbitone and pethidine on foetal breathing movements in sheep. 1 Small doses of pentobarbitone (4 mg/kg i.v.) administered to sheep in the last third of pregancy had little overt effect on the mothers. In the foetus they caused arrest of breathing movements, an alteration in the character of the electrocorticogram and cardiovascular changes which varied with gestational age. 2 In contrast, relatively large doses of pethidine (100-200 mg) admininstered to the mother had no consistent effect on normal foetal breathing movements, though they abolished the foetal response to hypercapnia. 3 The results are discussed in relation to feotal sleep state. | [
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PMID:7342 | Comparison of antihypertensive activity of beta-blocking drugs during chronic treatment. | The hypotensive activity of five beta-adrenoceptor antagonists with different ancillary pharmacological properties was compared in a randomised double-blind factorial trial in 25 untreated patients with stable, uncomplicated essential hypertension. In doses that produced similar reductions in exercise tachycardia all drugs had similar blood-pressure lowering activity, greater on systolic than diastolic pressure and greatest during exercise. With the exception of vasodilator activity the possession of any particular combinaton of ancillary pharmacological properties did not significantly influence the specific antihypertensive activity of these compounds. | Comparison of antihypertensive activity of beta-blocking drugs during chronic treatment. The hypotensive activity of five beta-adrenoceptor antagonists with different ancillary pharmacological properties was compared in a randomised double-blind factorial trial in 25 untreated patients with stable, uncomplicated essential hypertension. In doses that produced similar reductions in exercise tachycardia all drugs had similar blood-pressure lowering activity, greater on systolic than diastolic pressure and greatest during exercise. With the exception of vasodilator activity the possession of any particular combinaton of ancillary pharmacological properties did not significantly influence the specific antihypertensive activity of these compounds. | [
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PMID:7347 | Pain as a major cause of postoperative nausea. | The incidence of nausea in relation to pain was recorded in 104 patients after abdominal operations. Ten per cent of the patients had episodes of nausea not related to pain. One hundred and fourteen episodes of concomitant pain and nausea were recorded in 61 patients (58.6 per cent). The intravenous injection of morphine or ketobemidone relieved nausea as well as pain in 80 per cent of the episodes. Relief of pain with persistence of nausea was uncommon and if pain relief was inadequate nausea was unabated. Nausea was provoked by 3.4 per cent of the morphine injections, but all patients tolerated similar doses of morphine on other occasions without nausea. Nausea often accompanies pain in the early postoperative period and can be relieved concomitant with the pain by the intravenous use of opiates in adequate doses in a high proportion of cases. | Pain as a major cause of postoperative nausea. The incidence of nausea in relation to pain was recorded in 104 patients after abdominal operations. Ten per cent of the patients had episodes of nausea not related to pain. One hundred and fourteen episodes of concomitant pain and nausea were recorded in 61 patients (58.6 per cent). The intravenous injection of morphine or ketobemidone relieved nausea as well as pain in 80 per cent of the episodes. Relief of pain with persistence of nausea was uncommon and if pain relief was inadequate nausea was unabated. Nausea was provoked by 3.4 per cent of the morphine injections, but all patients tolerated similar doses of morphine on other occasions without nausea. Nausea often accompanies pain in the early postoperative period and can be relieved concomitant with the pain by the intravenous use of opiates in adequate doses in a high proportion of cases. | [
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PMID:7352 | Radiation-induced xerostomia in cancer patients. Effect on salivary and serum electrolytes. | Saliva and serum electrolyte concentrations were monitored in 30 patients given a course of xerostomia-producing cancer radiotherapy. The mean flow rate of stimulated whole saliva decreased 83.3% during a 6-week treatment period. The striking reduction in saliva output was accompanied by significant increases in saliva Na+, Cl-, Ca++, Mg++ and Prot.- concentrations and by a decrease in saliva HCO3- content. The xerostomic saliva was more concentrated and had a greater salinity than the pretreatment saliva in each instance. In contrast, none of the serum electrolytes measured was significantly altered by the subtotal salivary shutdown. | Radiation-induced xerostomia in cancer patients. Effect on salivary and serum electrolytes. Saliva and serum electrolyte concentrations were monitored in 30 patients given a course of xerostomia-producing cancer radiotherapy. The mean flow rate of stimulated whole saliva decreased 83.3% during a 6-week treatment period. The striking reduction in saliva output was accompanied by significant increases in saliva Na+, Cl-, Ca++, Mg++ and Prot.- concentrations and by a decrease in saliva HCO3- content. The xerostomic saliva was more concentrated and had a greater salinity than the pretreatment saliva in each instance. In contrast, none of the serum electrolytes measured was significantly altered by the subtotal salivary shutdown. | [
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PMID:7353 | Models for depolymerizing enzymes: criteria for discrimination of models. | Several models for the action of alpha amylase have been proposed to account for the nonrandom distribution of oligosaccharides in the amylase digests of polysaccharides. The preferred-attack model attempts to account for the nonrandom distribution by assuming that the probability for bond cleavage depends upon the position of the bond in the chain. The repetitive, or multiple-attack, model suggests that the nonrandom distribution of oligosaccharides arises because an amylase can form a cage-like complex with a substrate and attack it several times during a single encounter. The multiple-enzyme or dual-site model suggests that the nonrandom yield of oligosaccharides arises from the combined action of exo- and endo-enzymes. The effects of pH, inhibitors, and substrate chain-length on enzyme action have been studied in several laboratories to determine which of the three action-patterns best describes the action of alpha amylase. The influence of these variables on product distributions or enzyme action-patterns are mathematically modeled in the Appendix. The experimental data on porcine-pancreatic alpha amylase are discussed in the light of the derivations. | Models for depolymerizing enzymes: criteria for discrimination of models. Several models for the action of alpha amylase have been proposed to account for the nonrandom distribution of oligosaccharides in the amylase digests of polysaccharides. The preferred-attack model attempts to account for the nonrandom distribution by assuming that the probability for bond cleavage depends upon the position of the bond in the chain. The repetitive, or multiple-attack, model suggests that the nonrandom distribution of oligosaccharides arises because an amylase can form a cage-like complex with a substrate and attack it several times during a single encounter. The multiple-enzyme or dual-site model suggests that the nonrandom yield of oligosaccharides arises from the combined action of exo- and endo-enzymes. The effects of pH, inhibitors, and substrate chain-length on enzyme action have been studied in several laboratories to determine which of the three action-patterns best describes the action of alpha amylase. The influence of these variables on product distributions or enzyme action-patterns are mathematically modeled in the Appendix. The experimental data on porcine-pancreatic alpha amylase are discussed in the light of the derivations. | [
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PMID:7355 | Effects of angiographic contrast media on sino-atrial nodal function. | Injection of meglumine diatrizoate (Renografin-76) into the selectively perfused sinus node artery of the dog produces bradycardia which is unaltered by autonomic blockade or by changes in sinus node artery pressure. Contrast agents and other hyperosmolar substances prolong the R-R interval in proportion to their osmolarity. Selective injection of contrast media into other cannulated segments of the coronary tree produces no change in heart rate. Transfemoral arteriography, however, produces bradycardia with both right and left coronary injections. Both direct and reflex sinus node depression occur with coronary arteriography in the dog. Direct effects are mediated by hyperosmolarity. | Effects of angiographic contrast media on sino-atrial nodal function. Injection of meglumine diatrizoate (Renografin-76) into the selectively perfused sinus node artery of the dog produces bradycardia which is unaltered by autonomic blockade or by changes in sinus node artery pressure. Contrast agents and other hyperosmolar substances prolong the R-R interval in proportion to their osmolarity. Selective injection of contrast media into other cannulated segments of the coronary tree produces no change in heart rate. Transfemoral arteriography, however, produces bradycardia with both right and left coronary injections. Both direct and reflex sinus node depression occur with coronary arteriography in the dog. Direct effects are mediated by hyperosmolarity. | [
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PMID:7357 | The form and function of cnidarian spirocysts. 2. Ultrastructure of the capsule tip and wall and mechanism of discharge. | The electron-dense capsule tip (apical cap) of sea anemone and coral spirocysts is of a different structure than the capsule wall. The capsule wall is composed of a double layer of fiber-like materials which cross each other at roughly right angles. The innermost layer is characterized by numerous serrations, the tips of which project into the lumen of the capsule. Within each serration, a band of finely cross-striated material encircles the capsule at right angles to its longitudinal axis. The membrane lining the lumen of the capsule appears to be continuous with the wall of the undischarged thread. The outer capsule wall layer consists of closely spaced microfilaments (cnidofilaments) which are oriented in the longitudinal axis of the capsule. The cnidofilaments appear to merge with the apical cap material. Contrary to some previous reports in the literature, it has been found that spirocysts normally discharge by eversion, as do nematocysts. The relationship of the capsule wall sub-structure to the spirocyst discharge process is discussed. | The form and function of cnidarian spirocysts. 2. Ultrastructure of the capsule tip and wall and mechanism of discharge. The electron-dense capsule tip (apical cap) of sea anemone and coral spirocysts is of a different structure than the capsule wall. The capsule wall is composed of a double layer of fiber-like materials which cross each other at roughly right angles. The innermost layer is characterized by numerous serrations, the tips of which project into the lumen of the capsule. Within each serration, a band of finely cross-striated material encircles the capsule at right angles to its longitudinal axis. The membrane lining the lumen of the capsule appears to be continuous with the wall of the undischarged thread. The outer capsule wall layer consists of closely spaced microfilaments (cnidofilaments) which are oriented in the longitudinal axis of the capsule. The cnidofilaments appear to merge with the apical cap material. Contrary to some previous reports in the literature, it has been found that spirocysts normally discharge by eversion, as do nematocysts. The relationship of the capsule wall sub-structure to the spirocyst discharge process is discussed. | [
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PMID:7364 | Differential reaction of cell membrane phospholipids and proteins with chemical probes. | The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DFDNB) were used. The reaction of hemoglobin, membrane protein and membrane PE and PS of erythrocytes with DFNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. TNBS reacts to an extremely small extend with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS whereas the reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the erythrocyte membrane as compared to the outer surface. TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5-2.0 mM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5% of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPE from DNP-PE. A phosphodiesterase was also present which hydrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear, however, that TNBS and FDNB react to a different extent with PE in this cell. The Acholeplasma membrane had no detectable PE or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31% being on the outer surface and 69% on the inner surface... | Differential reaction of cell membrane phospholipids and proteins with chemical probes. The major aims of this study were to determine the degree of phospholipid asymmetry and the neighbor analysis of phospholipids in different types of cell membranes. For this study a penetrating probe (FDNB), a non-penetrating probe (TNBS) and a cross-linking probe (DFDNB) were used. The reaction of hemoglobin, membrane protein and membrane PE and PS of erythrocytes with DFNB and TNBS was studied over a concentration range of 0.5 to 10 mM probe. TNBS reacts to an extremely small extend with hemoglobin over the concentration range 0.4 to 4 mM whereas FDNB reacts with hemoglobin to a very large extent (50 fold more than TNBS). The reaction of membrane protein of intact erythrocytes reaches a sharp plateau at 1 mM TNBS whereas the reaction of membrane protein goes to a much larger extent with FDNB with no plateau seen up to 4 mM FDNB. This data shows that TNBS does not significantly penetrate into the cell under our conditions whereas FDNB does penetrate into the cell. The results show that there are four fold more reactive sites on proteins localized on the inner surface of the erythrocyte membrane as compared to the outer surface. TNBS at 0.5 to 2 mM concentration does not label membrane PS and labels membrane PE to a small extent. The reaction of PE with TNBS shows an initial plateau at 2 mM probe and a second slightly higher plateau between 4 to 10 mM probe. TNBS from 0.5-2.0 mM does not react with PS, but between 3 to 10 mM concentration, a very small amount of PS reacts with TNBS. Hence above 2 mM TNBS or FDNB a perturbation occurs in the membrane such that more PE and PS are exposed and react with these probes. These results demonstrate that essentially no PS is localized on the outer surface of the membrane and only 5% of the total membrane PE is localized on the outer surface of the erythrocyte membrane. TNBS and FDNB were reacted with yeast, E. coli, and Acholeplasma cells. With yeast cells, FDNB reacts to a much larger extent with PE than does TNBS, indicating that FDNB penetrates into the cell and labels more PE molecules. With E. coli, but not with erythrocytes or yeast cells, phospholipase A activity was very pronounced at pH 8.5 giving rise to a large amount of DNP-GPE from DNP-PE. A phosphodiesterase was also present which hydrolyized DNP-GPE to DNP-ethanolamine. The multilayered structure of the E. coli cell envelop did not permit a definitive interpretation of the results. It is clear, however, that TNBS and FDNB react to a different extent with PE in this cell. The Acholeplasma membrane had no detectable PE or PS but contains amino acid esters of phosphatidylglycerol. The reaction of these components with TNBS and FDNB indicate that these aminoacyl-PG are localized on both surfaces of the membrane, with 31% being on the outer surface and 69% on the inner surface... | [
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PMID:7367 | Simplified, totally enzymatic method for determination of serum triglycerides with a centrifugal analyzer. | We describe a totally enzymatic method for determination of serum triglycerides (triacylglycerols) specifically adaptable to the CentrifiChem system. The method involves lipolysis with lipase from Rhizopus arrhizus alone and quantitation of the resulting glycerol with glycerol dehydrogenase in a kinetic, fixed-time mode. Hydrolysis by the lipase is complete, for concentrations up to at least 5.0 g/liter, in 10 min at room temperature. The unfavorable equilibrium for the oxidation of glycerol is overcome by increasing the pH and adding excess NAD+. Under these conditions the glycerol determination is linear to at least 4.0 g of glycerol per liter, as triglyceride. The test exhibits acceptable accuracy and precision, and results correlate well with those by an alternative totally enzymatic procedure. The present method is unaffected by phosphatase and a considerably simplified reagent is used. | Simplified, totally enzymatic method for determination of serum triglycerides with a centrifugal analyzer. We describe a totally enzymatic method for determination of serum triglycerides (triacylglycerols) specifically adaptable to the CentrifiChem system. The method involves lipolysis with lipase from Rhizopus arrhizus alone and quantitation of the resulting glycerol with glycerol dehydrogenase in a kinetic, fixed-time mode. Hydrolysis by the lipase is complete, for concentrations up to at least 5.0 g/liter, in 10 min at room temperature. The unfavorable equilibrium for the oxidation of glycerol is overcome by increasing the pH and adding excess NAD+. Under these conditions the glycerol determination is linear to at least 4.0 g of glycerol per liter, as triglyceride. The test exhibits acceptable accuracy and precision, and results correlate well with those by an alternative totally enzymatic procedure. The present method is unaffected by phosphatase and a considerably simplified reagent is used. | [
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PMID:7368 | Measuring water content of feces by the Karl Fischer method. | We describe a technique for measuring the water content of stools by the Karl Fischer method. The analysis is based on removal of water into a mixture of methanol/chloroform (1/2), after dispersion of the stool by sonication in presence of solvent. An aliquot of the solution thus obtained is placed in themeasuring cell of a Karl Fischer apparatus and then analyzed in the classic way. We further describe the advantages of this method (odorless, precise, reproducible) in contrast to other current methods. In addition the same organic solution can also be used in determining the lipid content of stools. | Measuring water content of feces by the Karl Fischer method. We describe a technique for measuring the water content of stools by the Karl Fischer method. The analysis is based on removal of water into a mixture of methanol/chloroform (1/2), after dispersion of the stool by sonication in presence of solvent. An aliquot of the solution thus obtained is placed in themeasuring cell of a Karl Fischer apparatus and then analyzed in the classic way. We further describe the advantages of this method (odorless, precise, reproducible) in contrast to other current methods. In addition the same organic solution can also be used in determining the lipid content of stools. | [
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PMID:7369 | Stability and precision of a new ampuled quality-control system for pH and blood-gas measurements. | We describe an evaluation of the in-use stability and short-term precision of a three-level ampuled quality-control system for monitoring pH, pCO2, and pO2 measurements on clinical blood-gas analyzers. In three hospital laboratories, 324 such ampuls were opened and allowed to stand with their contents exposed to atmospheric conditions for accurately timed intervals up to 240 s. Contents were then analyzed for pH, pCO2, and pO2. Student's t-test was used to evaluate the significance of differences observed in recoveries after time exposure. At a signifcance level of P less than or equal to 0.05, the only significant changes observed throughout the first minute of exposure were average pO2 increases of 180 Pa (1.4 mmHg) (+ 1.4%) and 230 Pa (1.7 mmHg) (+ 2.9%) at levels of 13.4 and 7.7 kPa kPa (101 and 58 mmHg), respectively. The ampuled system was found to be stable precise convenient, and suitable for use in the routine laboratory. | Stability and precision of a new ampuled quality-control system for pH and blood-gas measurements. We describe an evaluation of the in-use stability and short-term precision of a three-level ampuled quality-control system for monitoring pH, pCO2, and pO2 measurements on clinical blood-gas analyzers. In three hospital laboratories, 324 such ampuls were opened and allowed to stand with their contents exposed to atmospheric conditions for accurately timed intervals up to 240 s. Contents were then analyzed for pH, pCO2, and pO2. Student's t-test was used to evaluate the significance of differences observed in recoveries after time exposure. At a signifcance level of P less than or equal to 0.05, the only significant changes observed throughout the first minute of exposure were average pO2 increases of 180 Pa (1.4 mmHg) (+ 1.4%) and 230 Pa (1.7 mmHg) (+ 2.9%) at levels of 13.4 and 7.7 kPa kPa (101 and 58 mmHg), respectively. The ampuled system was found to be stable precise convenient, and suitable for use in the routine laboratory. | [
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