Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.4.1.1 (pyruvate carboxylase)
 1,516 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.
Biochem J 1999 Sep 15
PMID:Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study. 1047 67

The glucose-fatty acid cycle of Randle entails two elements: decreased pyruvate dehydrogenase (PDH) activity, which inhibits glucose oxidation, and inhibition of phosphofructokinase (PFK) by a rise in citrate so that glucose-6-phosphate (G-6-P) levels increase, thereby inhibiting hexokinase activity and hence glucose utilization. Chronic exposure of islets to long-chain fatty acids (FA) is reported to lower PDH activity, but the effect on glucose oxidation and glucose-induced insulin secretion is uncertain. We investigated rat islets that were cultured for 4 days with 0.25 mmol/l oleate/5.5 mmol/l glucose. Glucose oxidation was doubled at 2.8 mmol/l glucose and unchanged at 27.7 mmol/l glucose in the FA-cultured islets despite a 35% decrease in assayed PDH activity. Pyruvate content was increased 60%, which may well compensate for the decreased PDH activity and maintain flux through the citric acid cycle. However, a greater diversion of pyruvate metabolism through the pyruvate-malate shuttle is suggested by unchanged pyruvate carboxylase Vmax and a fourfold higher release of malate from isolated mitochondria. The FA-cultured islets also showed increased basal glucose usage and insulin secretion together with a lowered level of G-6-P and 50% reductions in citrate synthase Vmax and the citrate content. Thus, the effects of chronic FA exposure on islet glucose metabolism differ from the glucose-fatty acid interactions reported in some other tissues.
Diabetes 1999 Sep
PMID:Glucose-fatty acid cycle to inhibit glucose utilization and oxidation is not operative in fatty acid-cultured islets. 1048 Jun 4

The 13C-label incorporation into glutamate, glutamine, aspartate and gamma-aminobutyric acid (GABA) from [2-13C] glucose was measured by 13C nuclear magnetic resonance (NMR) spectroscopy to directly examine the effects of ammonia on the activity of pyruvate carboxylase (i.e., the anaplerotic pathway) and the amino acid metabolism in the rat brain in vivo. Rats were sacrificed by exposure to microwaves at 7.5, 15, 30, and 60 min after an i.v. injection of [2-13C] glucose with or without ammonium acetate. After the injection of ammonium acetate, the brain contents of glutamate, aspartate and GABA had decreased, however, the percentage of 13C enrichment of C3 of glutamine, glutamate and GABA, and C2 and C3 of aspartate had increased. The 13C entered the TCA cycle via pyruvate carboxylase from [2-13C] glucose, labeling the C2 or C3 positions of aspartate, the C2 or C3 positions of glutamate and glutamine, and the C3 or C4 positions of GABA first and second turns of the tricarboxylic acid (TCA) cycle. The C4/C3 labeling ratio in GABA was lower than the analogous ratio in glutamate (C2/C3) and higher than that of glutamine (C2/C3). The order of these ratios (glutamate > GABA > glutamine) was not altered by the injection of ammonium acetate. These findings directly indicate that ammonia increases the anaplerotic pathway and that the 13C-skeletons entered glial glutamine through the anaplerotic pathway flow from glia to neuron. A fraction of the glutamine is used in the direct synthesis of GABA via glutamate, whereas the remaining fraction of glutamine passed through the neuronal TCA cycle before synthesizing GABA.
Brain Res 1999 Sep 11
PMID:Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo 13C NMR spectroscopic study of rats after administering [2-13C]] glucose with or without ammonium acetate. 1054 83

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal C2-lambda phage genomic library. Downstream of the pyruvate carboxylase gene-containing fragment, a gene cluster coding for open reading frames displaying extensive homology to citrate synthase, aconitase, and a truncated isocitrate dehydrogenase was identified. However, the truncation was shown to have occurred during the cloning by two noncontiguous Sau3AI fragments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lactococcal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Corresponding to the complete citrate synthase gene, citrate synthase activity was detected in Lactococcus lactis ssp. lactis C2. Isocitrate dehydrogenase activity was found to be missing in Lactococcus lactis C2, suggesting that the gene may be incomplete or is not expressed, resulting in a requirement for glutamic acid in lactococci.
J Dairy Sci 2000 Sep
PMID:Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp. lactis C2. 1100 18

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.
Anal Biochem 2001 Sep 15
PMID:Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? 1155 13

We have cloned and sequenced the gene encoding mouse pyruvate carboxylase (mPC) [EC 6.4.1.1]. The coding region contains 19 exons, one 5'-untranslated region exon, and 19 introns in 22 kb of genomic DNA. This gene's exon/intron organization is highly conserved with respect to rat and human PC genes. The mPC gene promoter lacks canonical TATA and CCAAT boxes, in common with a number of housekeeping genes. Transient expressions in COS-1 of a luciferase reporter gene under the control of 5'-nested deletions of the 5'-flanking sequence of the mPC gene have identified the 166-bp minimal sequence required for basal transcription. Alternative splicing at the 5'-untranslated region exon of the mouse PC gene results in the production of two alternate transcripts bearing different 5'-noncoding regions. Both transcripts are highly expressed in kidney and liver and moderately expressed in heart and testis and expressed at a low level in spleen.
Biochem Biophys Res Commun 2001 Sep 21
PMID:Structural and promoter regions of the murine pyruvate carboxylase gene. 1155 43

Nutrient secretagogues can increase the production of succinyl-CoA in rat pancreatic islets. When succinate esters are the secretagogue, succinyl-CoA can be generated via the succinate thiokinase reaction. Other secretagogues can increase production of succinyl-CoA secondary to increasing alpha-ketoglutarate production by glutamate dehydrogenase or mitochondrial aspartate aminotransferase followed by the alpha-ketoglutarate dehydrogenase reaction. Although secretagogues can increase the production of succinyl-CoA, they do not increase the level of this metabolite until after they decrease the level of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This suggests that the generated succinyl-CoA initially reacts with acetoacetate to yield acetoacetyl-CoA plus succinate in the succinyl-CoA-acetoacetate transferase reaction. This would be followed by acetoacetyl-CoA reacting with acetyl-CoA to generate HMG-CoA in the HMG-CoA synthetase reaction. HMG-CoA will then be reduced by NADPH to mevalonate in the HMG-CoA reductase reaction and/or cleaved to acetoacetate plus acetyl-CoA by HMG cleavage enzyme. Succinate derived from either exogenous succinate esters or generated by succinyl-CoA-acetoacetate transferase is metabolized to malate followed by the malic enzyme reaction. Increased production of NADPH by the latter reaction then increases reduction of HMG-CoA and accounts for the decrease in the level of HMG-CoA produced by secretagogues. Pyruvate carboxylation catalyzed by pyruvate carboxylase will supply oxaloacetate to mitochondrial aspartate aminotransferase. This would enable this aminotransferase to supply alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex and would, in part, account for secretagogues increasing the islet level of succinyl-CoA after they decrease the level of HMG-CoA. Mevalonate could be a trigger of insulin release as a result of its ability to alter membrane proteins and/or cytosolic Ca(2+). This is consistent with the fact that insulin secretagogues decrease the level of the mevalonate precursor HMG-CoA. In addition, inhibitors of HMG-CoA reductase interfere with insulin release and this inhibition can be reversed by mevalonate.
Diabetes 2002 Sep
PMID:The succinate mechanism of insulin release. 1219 57

Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-beta-hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene ( pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly-beta-hydroxybutyrate.
Appl Microbiol Biotechnol 2004 Sep
PMID:Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-beta-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium. 1512 63

Transcarboxylase is a 1.2 million Dalton (Da) multienzyme complex from Propionibacterium shermanii that couples two carboxylation reactions, transferring CO(2)(-) from methylmalonyl-CoA to pyruvate to yield propionyl-CoA and oxaloacetate. Crystal structures of the 5S metalloenzyme subunit, which catalyzes the second carboxylation reaction, have been solved in free form and bound to its substrate pyruvate, product oxaloacetate, or inhibitor 2-ketobutyrate. The structure reveals a dimer of beta(8)alpha(8) barrels with an active site cobalt ion coordinated by a carbamylated lysine, except in the oxaloacetate complex in which the product's carboxylate group serves as a ligand instead. 5S and human pyruvate carboxylase (PC), an enzyme crucial to gluconeogenesis, catalyze similar reactions. A 5S-based homology model of the PC carboxyltransferase domain indicates a conserved mechanism and explains the molecular basis of mutations in lactic acidemia. PC disease mutations reproduced in 5S result in a similar decrease in carboxyltransferase activity and crystal structures with altered active sites.
EMBO J 2004 Sep 15
PMID:Transcarboxylase 5S structures: assembly and catalytic mechanism of a multienzyme complex subunit. 1532 73

In addition to lactate and pyruvate, some amino acids were found to serve as potential gluconeogenic substrates in the perfused liver of Clarias batrachus. Glutamate was found to be the most effective substrate, followed by lactate, pyruvate, serine, ornithine, proline, glutamine, glycine, and aspartate. Four gluconeogenic enzymes, namely phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase (PC), fructose 1,6-bisphosphatase (FBPase) and glucose 6-phosphatase (G6Pase) could be detected mainly in liver and kidney, suggesting that the latter are the two major organs responsible for gluconeogenic activity in this fish. Hypo-osmotically induced cell swelling caused a significant decrease of gluconeogenic efflux accompanied with significant decrease of activities of PEPCK, FBPase and G6Pase enzymes in the perfused liver. Opposing effects were seen in response to hyperosmotically induced cell shrinkage. These changes were partly blocked in the presence of cycloheximide, suggesting that the aniso-osmotic regulations of gluconeogenesis possibly occurs through an inverse regulation of enzyme proteins and/or a regulatory protein synthesis in this catfish. In conclusion, gluconeogenesis appears to play a vital role in C. batrachus in maintaining glucose homeostasis, which is influenced by cell volume changes possibly for proper energy supply under osmotic stress.
J Biosci 2004 Sep
PMID:Cell volume changes affect gluconeogenesis in the perfused liver of the catfish Clarias batrachus. 1538 55


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