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John C. Wallace - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms of inhibition of Rhizobium etli Pyruvate Carboxylase by L-aspartate.
Biochemistry, 2014Co-Authors: Chaiyos Sirithanakorn, John C. Wallace, Abdussalam Adina-zada, Sarawut Jitrapakdee, Paul V. AttwoodAbstract:l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme Pyruvate Carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of Pyruvate Carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli Pyruvate Carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxama...
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Kinetic characterization of yeast Pyruvate Carboxylase isozyme pyc1.
Biochemistry, 2002Co-Authors: Joy P. Branson, John C. Wallace, Mark Nezic, Paul V. AttwoodAbstract:Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for Pyruvate Carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast Pyruvate Carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the Ka of this isozyme for acetyl CoA is in the order of 8−10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver Pyruvate Carboxylase. However, unlike the chicken liver enzyme, the...
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Yeast Pyruvate Carboxylase: identification of two genes encoding isoenzymes.
Biochemical and biophysical research communications, 1991Co-Authors: Michelle E. Walker, Manfred Rohde, Dale L. Val, Rodney J. Devenish, John C. WallaceAbstract:In Saccharomyces cerevisiae, Pyruvate Carboxylase [EC 6.4.1.1] has an important anaplerotic role in the production of oxaloacetate from Pyruvate. We report here the existence of two Pyruvate Carboxylase isozymes, which are encoded by separate genes within the yeast genome. Null mutants were constructed by one step gene disruption of the characterised PYC gene in the yeast genome. The mutants were found to have 10-20% residual Pyruvate Carboxylase activity, which was attributable to a protein of identical size and immunogenically related to Pyruvate Carboxylase. Immunocytochemical labelling studies on ultrathin sections of embedded whole cells from the null mutants showed the isozyme to be located exclusively in the cytoplasm. We have mapped the genes encoding both enzymes and shown the previously characterised gene, designated PYC1, to be on chromosome VII whilst PYC2 is on chromosome II.
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Isolation of a yeast mutant deficient in Pyruvate Carboxylase activity.
Biochemistry international, 1991Co-Authors: Michelle E. Walker, John C. WallaceAbstract:To improve our understanding of the catalytic mechanism and regulatory properties of Pyruvate Carboxylase (EC 6.4.1.1), an important biotin-dependent enzyme, we have sought to isolate mutants in Saccharomyces cerevisiae which are defective in Pyruvate Carboxylase activity. One mutant was isolated which was unable to grow on glucose minimal medium unless supplemented with aspartate. Although the enzyme had only 25% of the wild type Pyruvate Carboxylase activity, Western analysis and RNase protection analysis demonstrated that the mutant gene was expressed at approximately 70% of the wild type level. On the basis of genetic crosses and complementation tests, we have attributed the defect to mutations in the PYC gene encoding Pyruvate Carboxylase.
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Electron microscopic localization of Pyruvate Carboxylase in rat liver and Saccharomyces cerevisiae by immunogold procedures
Archives of biochemistry and biophysics, 1991Co-Authors: Manfred Rohde, Filip Lim, John C. WallaceAbstract:Abstract The intracellular location of Pyruvate Carboxylase (EC 6.4.1.1) in rat liver and Saccharomyces cerevisiae was investigated using the antibody-gold and protein A-gold techniques carried out as a postembedding immunoelectron microscopic procedure. The vast majority of gold particles (> 98%), indicative of the presence of antigenic sites of Pyruvate Carboxylase, were found in the mitochondria of rat liver. No other cellular compartment was labeled except the cytosol which did not account for more than 2% of the total labeling of a rat hepatocyte. Furthermore, 60% of labeled Pyruvate Carboxylase molecules within a mitochondrion were found adjacent to the matrix side of the inner mitochondrial membrane. In contrast, in S. cerevisiae, Pyruvate Carboxylase was found exclusively in the cytosol.
Paul V. Attwood - One of the best experts on this subject based on the ideXlab platform.
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Mechanisms of inhibition of Rhizobium etli Pyruvate Carboxylase by L-aspartate.
Biochemistry, 2014Co-Authors: Chaiyos Sirithanakorn, John C. Wallace, Abdussalam Adina-zada, Sarawut Jitrapakdee, Paul V. AttwoodAbstract:l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme Pyruvate Carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of Pyruvate Carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli Pyruvate Carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxama...
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Inhibitors of Pyruvate Carboxylase
The open enzyme inhibition journal, 2010Co-Authors: Tonya N. Zeczycki, Martin St. Maurice, Paul V. AttwoodAbstract:This review aims to discuss the varied types of inhibitors of biotin-dependent Carboxylases, with an emphasis on the inhibitors of Pyruvate Carboxylase. Some of these inhibitors are physiologically relevant, in that they provide ways of regulating the cellular activities of the enzymes e.g. aspartate and prohibitin inhibition of Pyruvate Carboxylase. Most of the inhibitors that will be discussed have been used to probe various aspects of the structure and function of these enzymes. They target particular parts of the structure e.g. avidin - biotin, FTP - ATP binding site, oxamate - Pyruvate binding site, phosphonoacetate - binding site of the putative carboxyphosphate intermediate.
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Kinetic characterization of yeast Pyruvate Carboxylase isozyme pyc1.
Biochemistry, 2002Co-Authors: Joy P. Branson, John C. Wallace, Mark Nezic, Paul V. AttwoodAbstract:Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for Pyruvate Carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast Pyruvate Carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the Ka of this isozyme for acetyl CoA is in the order of 8−10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver Pyruvate Carboxylase. However, unlike the chicken liver enzyme, the...
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88 Cysteine-lysine ion pairs in yeast Pyruvate Carboxylase
Biochemical Society transactions, 1998Co-Authors: Madeleine J. Headlam, Paul V. AttwoodAbstract:Pyruvate carboxylasc [EC 6.4.1.11 catalyses the carboxylation of Pyruvate to produce oxaloacetate. The overall reaction catalysed by Pyruvate Carboxylase and other biotindependent enzymes is thought to occur in two partial reactions at spatially separate subsites (for review see [ 11). The first partial reaction of Pyruvate Carboxylase requires carboxylation of the biotin prosthetic p u p , eq.(l) and the second partial reaction involves carboxylation of Pyruvate,
Kevin L Behar - One of the best experts on this subject based on the ideXlab platform.
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cerebral Pyruvate Carboxylase flux is unaltered during bicuculline seizures
Journal of Neuroscience Research, 2005Co-Authors: Anant B Patel, Golam M I Chowdhury, Robin A De Graaf, Douglas L Rothman, Robert G Shulman, Kevin L BeharAbstract:Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and γ-aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by Pyruvate Carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between Pyruvate Carboxylase flux and neuronal activity is not known. In this study, Pyruvate Carboxylase flux was assessed during intravenous infusion of [2- 1 3 C]glucose using localized 1 H-[ 1 3 C] NMR spectroscopy at 7 Tesla in vivo in halothane-anesthetized and ventilated adult Wistar rats during 85 min of bicuculline-induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate Carboxylase flux was assessed by the sum of [2- 1 3 C] and [3- 1 3 C] of glutamine and glutamate (Glx 2 + 3 ) labeling during [2- 1 3 C]glucose infusion. During seizures the initial rate of Glx 2 + 3 synthesis (0.069 ′ 0.013 μmol/ g/min) was not significantly different (P = 0.68) from that of the controls (0.059 ′ 0.010 μmol/g/min), indicating that anaplerotic flow through Pyruvate Carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through Pyruvate Carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b).
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Cerebral Pyruvate Carboxylase flux is unaltered during bicuculline‐seizures
Journal of Neuroscience Research, 2004Co-Authors: Anant B Patel, Golam M I Chowdhury, Robin A De Graaf, Douglas L Rothman, Robert G Shulman, Kevin L BeharAbstract:Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and γ-aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by Pyruvate Carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between Pyruvate Carboxylase flux and neuronal activity is not known. In this study, Pyruvate Carboxylase flux was assessed during intravenous infusion of [2- 1 3 C]glucose using localized 1 H-[ 1 3 C] NMR spectroscopy at 7 Tesla in vivo in halothane-anesthetized and ventilated adult Wistar rats during 85 min of bicuculline-induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate Carboxylase flux was assessed by the sum of [2- 1 3 C] and [3- 1 3 C] of glutamine and glutamate (Glx 2 + 3 ) labeling during [2- 1 3 C]glucose infusion. During seizures the initial rate of Glx 2 + 3 synthesis (0.069 ′ 0.013 μmol/ g/min) was not significantly different (P = 0.68) from that of the controls (0.059 ′ 0.010 μmol/g/min), indicating that anaplerotic flow through Pyruvate Carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through Pyruvate Carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b).
Manfred Rohde - One of the best experts on this subject based on the ideXlab platform.
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Yeast Pyruvate Carboxylase: identification of two genes encoding isoenzymes.
Biochemical and biophysical research communications, 1991Co-Authors: Michelle E. Walker, Manfred Rohde, Dale L. Val, Rodney J. Devenish, John C. WallaceAbstract:In Saccharomyces cerevisiae, Pyruvate Carboxylase [EC 6.4.1.1] has an important anaplerotic role in the production of oxaloacetate from Pyruvate. We report here the existence of two Pyruvate Carboxylase isozymes, which are encoded by separate genes within the yeast genome. Null mutants were constructed by one step gene disruption of the characterised PYC gene in the yeast genome. The mutants were found to have 10-20% residual Pyruvate Carboxylase activity, which was attributable to a protein of identical size and immunogenically related to Pyruvate Carboxylase. Immunocytochemical labelling studies on ultrathin sections of embedded whole cells from the null mutants showed the isozyme to be located exclusively in the cytoplasm. We have mapped the genes encoding both enzymes and shown the previously characterised gene, designated PYC1, to be on chromosome VII whilst PYC2 is on chromosome II.
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Electron microscopic localization of Pyruvate Carboxylase in rat liver and Saccharomyces cerevisiae by immunogold procedures
Archives of biochemistry and biophysics, 1991Co-Authors: Manfred Rohde, Filip Lim, John C. WallaceAbstract:Abstract The intracellular location of Pyruvate Carboxylase (EC 6.4.1.1) in rat liver and Saccharomyces cerevisiae was investigated using the antibody-gold and protein A-gold techniques carried out as a postembedding immunoelectron microscopic procedure. The vast majority of gold particles (> 98%), indicative of the presence of antigenic sites of Pyruvate Carboxylase, were found in the mitochondria of rat liver. No other cellular compartment was labeled except the cytosol which did not account for more than 2% of the total labeling of a rat hepatocyte. Furthermore, 60% of labeled Pyruvate Carboxylase molecules within a mitochondrion were found adjacent to the matrix side of the inner mitochondrial membrane. In contrast, in S. cerevisiae, Pyruvate Carboxylase was found exclusively in the cytosol.
Anant B Patel - One of the best experts on this subject based on the ideXlab platform.
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cerebral Pyruvate Carboxylase flux is unaltered during bicuculline seizures
Journal of Neuroscience Research, 2005Co-Authors: Anant B Patel, Golam M I Chowdhury, Robin A De Graaf, Douglas L Rothman, Robert G Shulman, Kevin L BeharAbstract:Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and γ-aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by Pyruvate Carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between Pyruvate Carboxylase flux and neuronal activity is not known. In this study, Pyruvate Carboxylase flux was assessed during intravenous infusion of [2- 1 3 C]glucose using localized 1 H-[ 1 3 C] NMR spectroscopy at 7 Tesla in vivo in halothane-anesthetized and ventilated adult Wistar rats during 85 min of bicuculline-induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate Carboxylase flux was assessed by the sum of [2- 1 3 C] and [3- 1 3 C] of glutamine and glutamate (Glx 2 + 3 ) labeling during [2- 1 3 C]glucose infusion. During seizures the initial rate of Glx 2 + 3 synthesis (0.069 ′ 0.013 μmol/ g/min) was not significantly different (P = 0.68) from that of the controls (0.059 ′ 0.010 μmol/g/min), indicating that anaplerotic flow through Pyruvate Carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through Pyruvate Carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b).
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Cerebral Pyruvate Carboxylase flux is unaltered during bicuculline‐seizures
Journal of Neuroscience Research, 2004Co-Authors: Anant B Patel, Golam M I Chowdhury, Robin A De Graaf, Douglas L Rothman, Robert G Shulman, Kevin L BeharAbstract:Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and γ-aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by Pyruvate Carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between Pyruvate Carboxylase flux and neuronal activity is not known. In this study, Pyruvate Carboxylase flux was assessed during intravenous infusion of [2- 1 3 C]glucose using localized 1 H-[ 1 3 C] NMR spectroscopy at 7 Tesla in vivo in halothane-anesthetized and ventilated adult Wistar rats during 85 min of bicuculline-induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate Carboxylase flux was assessed by the sum of [2- 1 3 C] and [3- 1 3 C] of glutamine and glutamate (Glx 2 + 3 ) labeling during [2- 1 3 C]glucose infusion. During seizures the initial rate of Glx 2 + 3 synthesis (0.069 ′ 0.013 μmol/ g/min) was not significantly different (P = 0.68) from that of the controls (0.059 ′ 0.010 μmol/g/min), indicating that anaplerotic flow through Pyruvate Carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through Pyruvate Carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b).
