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Chandra Mohan - One of the best experts on this subject based on the ideXlab platform.
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impaired mitochondrial metabolism and reduced Amphibolic krebs cycle activity in diabetic rat hepatocytes
Iubmb Life, 1995Co-Authors: R A Memon, Samuel P. Bessman, Chandra MohanAbstract:Oxidation of [2,3(14)C] and [1,4(14)C] succinate carbons in the mitochondrial Krebs cycle (KC) was used as a probe to investigate the effect of insulin and diabetes on mitochondrial metabolism in isolated rat hepatocytes. The data presented show that mitochondrial oxidation of succinate carbons and their incorporation into protein and lipid was markedly lower in diabetic and insulin treated diabetic rat hepatocytes. Unlike controls, diabetic rat hepatocytes were unresponsive to in vitro insulin addition. Amphibolic channeling of [2,3(14)C] succinate carbons into amino acid fraction was reduced in hepatocytes from diabetic rats, however, more of these carbons were diverted into the gluconeogenesis pathway. These data suggest that the diminished level of anabolic activities in the diabetic rat hepatocytes may be due to impairment in the KC reactions and a subsequent reduction in Amphibolic channeling of metabolic intermediates.
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Amphibolic role of the Krebs cycle in the insulin-stimulated protein synthesis
Archives of Biochemistry and Biophysics, 1991Co-Authors: Chandra Mohan, Riaz A. Memon, Samuel P. BessmanAbstract:Abstract It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14CO2 formation and protein incorporation of several 1-14C-labeled or U-14C-labeled amino acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced 14CO2 formation and protein incorporation primarily of those carbons of amino acids which are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 m m ), a potent inhibitor of the transamination reaction, it was shown that there exists an “insulin-sensitive” pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of 14C-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitochondrial transamination of α-ketoglutarate to glutamate is essential for insulin stimulation of 14C incorporation into hepatocyte protein.
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Ammonia inhibits insulin stimulation of the Krebs cycle: further insight into mechanism of hepatic coma.
Neurochemical Research, 1991Co-Authors: Samuel P. Bessman, Wei Wang, Chandra MohanAbstract:Oxidation of [2,3-14C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in14CO2 formation from [2,3-14C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the Amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased Amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.
Claus Schnarrenberger - One of the best experts on this subject based on the ideXlab platform.
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microsequecing and cdna cloning of the calvin cycle oppp enzyme ribose 5 phosphate isomerase ec 5 3 1 6 from spinach chloroplasts
Plant Molecular Biology, 1996Co-Authors: William Martin, Katrin Henze, Anke Flechner, Josef Kellermann, Claus SchnarrenbergerAbstract:Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single Amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.
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Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves.
Plant Physiology, 1995Co-Authors: Claus Schnarrenberger, Anke Flechner, William MartinAbstract:The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be Amphibolic for the Calvin cycle and OPPP.
Samuel P. Bessman - One of the best experts on this subject based on the ideXlab platform.
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impaired mitochondrial metabolism and reduced Amphibolic krebs cycle activity in diabetic rat hepatocytes
Iubmb Life, 1995Co-Authors: R A Memon, Samuel P. Bessman, Chandra MohanAbstract:Oxidation of [2,3(14)C] and [1,4(14)C] succinate carbons in the mitochondrial Krebs cycle (KC) was used as a probe to investigate the effect of insulin and diabetes on mitochondrial metabolism in isolated rat hepatocytes. The data presented show that mitochondrial oxidation of succinate carbons and their incorporation into protein and lipid was markedly lower in diabetic and insulin treated diabetic rat hepatocytes. Unlike controls, diabetic rat hepatocytes were unresponsive to in vitro insulin addition. Amphibolic channeling of [2,3(14)C] succinate carbons into amino acid fraction was reduced in hepatocytes from diabetic rats, however, more of these carbons were diverted into the gluconeogenesis pathway. These data suggest that the diminished level of anabolic activities in the diabetic rat hepatocytes may be due to impairment in the KC reactions and a subsequent reduction in Amphibolic channeling of metabolic intermediates.
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Amphibolic role of the Krebs cycle in the insulin-stimulated protein synthesis
Archives of Biochemistry and Biophysics, 1991Co-Authors: Chandra Mohan, Riaz A. Memon, Samuel P. BessmanAbstract:Abstract It has been a generally held view that insulin does not significantly affect the incorporation of amino acids into liver protein. This interpretation was based on data obtained from studies using the branched chain amino acids, which are poorly metabolized by the hepatic tissue. The effect of insulin on 14CO2 formation and protein incorporation of several 1-14C-labeled or U-14C-labeled amino acids was studied in isolated rat hepatocytes and diaphragm pieces. It was shown that insulin enhanced 14CO2 formation and protein incorporation primarily of those carbons of amino acids which are metabolized through the mitochondrial Krebs cycle. Using aminooxyacetic acid (0.5 m m ), a potent inhibitor of the transamination reaction, it was shown that there exists an “insulin-sensitive” pool of glutamate which is preferentially utilized for protein synthesis in the presence of insulin. The insulin effect on protein incorporation of 14C-labeled glutamate generated in the Krebs cycle was abolished in the presence of aminooxyacetic acid. We interpret these results to signify that mitochondrial transamination of α-ketoglutarate to glutamate is essential for insulin stimulation of 14C incorporation into hepatocyte protein.
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Ammonia inhibits insulin stimulation of the Krebs cycle: further insight into mechanism of hepatic coma.
Neurochemical Research, 1991Co-Authors: Samuel P. Bessman, Wei Wang, Chandra MohanAbstract:Oxidation of [2,3-14C]succinate in the intramitochondrial Krebs cycle was used as a probe to investigate the effect of ammonia on protein incorporation and Krebs cycle oxidation of succinate carbons in isolated rat hepatocytes. At low concentrations of ammonium chloride (0.1 to 0.5 mM) a slight increase in14CO2 formation from [2,3-14C]succinate was observed, however, the stimulatory effect of insulin was significantly reduced. Insulin failed to cause any stimulation of succinate carbons incorporation into hepatocyte protein in the presence of ammonium chloride. Addition of ammonium chloride also depressed the movement of tracer carbons into the gluconeogenesis pathway. The activity of the Amphibolic amino acid pool was significantly enhanced by ammonia. The data presented in this paper lend strong support to the Krebs-cycle depletion theory of hepatic coma. They also suggest that reduced mitochondrial Krebs cycle activity caused by increased Amphibolic depletion of substrates results in loss of insulin sensitivity in ammonia toxicity.
William Martin - One of the best experts on this subject based on the ideXlab platform.
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microsequecing and cdna cloning of the calvin cycle oppp enzyme ribose 5 phosphate isomerase ec 5 3 1 6 from spinach chloroplasts
Plant Molecular Biology, 1996Co-Authors: William Martin, Katrin Henze, Anke Flechner, Josef Kellermann, Claus SchnarrenbergerAbstract:Ribose-5-phosphate isomerase (RPI) catalyses the interconversion of ribose-5-phosphate and ribulose-5-phosphate in the reductive and oxidative pentose phosphate pathways in plants. RPI from spinach chloroplasts was purified and microsequenced. Via PCR with degenerate primers designed against microsequenced peptides, a hybridisation probe was obtained and used to isolate several cDNA clones which encode RPI. The nuclear-encoded 239 amino acid mature RPI subunit has a predicted size of 25.3 kDa and is translated as a cytosolic precursor possessing a 50 amino acid transit peptide. The processing site of the transit peptide was identified from protein sequence data. Spinach leaves possess only one type of homodimeric RPI enzyme which is localized in chloroplasts and is encoded by a single nuclear gene. Molecular characterization of RPI supports the view that a single Amphibolic RPI enzyme functions in the oxidative and reductive pentose phosphate pathways of spinach plastids.
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Enzymatic Evidence for a Complete Oxidative Pentose Phosphate Pathway in Chloroplasts and an Incomplete Pathway in the Cytosol of Spinach Leaves.
Plant Physiology, 1995Co-Authors: Claus Schnarrenberger, Anke Flechner, William MartinAbstract:The intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea L.) leaves. We found highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate path-way (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. The chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be Amphibolic for the Calvin cycle and OPPP.
William Marti - One of the best experts on this subject based on the ideXlab platform.
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cloning of the Amphibolic calvin cycle oppp enzyme d ribulose 5 phosphate 3 epimerase ec 5 1 3 1 from spinach chloroplasts functional and evolutionary aspects
Plant Molecular Biology, 1995Co-Authors: Ulrich Nowitzki, Claus Schnarrenberge, Pete Westhoff, Katri Henze, Ralf Wyrich, William MartiAbstract:Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T7 promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.