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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)

The 2' , 3'-dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg-oATP2) was shown to ba linear competitive inhibitor with respect to MgATP2-in both the acetyl-CoA-dependent and -independent activities of the enzyme but was a non-competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg-oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme...
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PMID:Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site. 124 74

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.
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PMID:The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment. 137 Apr 47

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.
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PMID:The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. 137 Apr 69

When chicken liver pyruvate carboxylase was incubated with either H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.
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PMID:Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction. 139 Jul 26

Veillonella parvula cannot grow with succinate as sole energy source. However, succinate decarboxylation simultaneous with malate or lactate fermentation increased growth yields by 2.4-3.5 g (mol succinate)-1. Malate was fermented stoichiometrically to acetate and propionate whereas lactate fermentation produced more acetate and considerable amounts of H2. Aspartate was utilized only in the presence of succinate as co-substrate. Methylmalonyl-CoA decarboxylase and ATP-dependent pyruvate carboxylase, but not methylmalonyl-CoA:pyruvate transcarboxylase, were detected in cell-free extracts of malate- or lactate-grown cells. The energetic aspects of these fermentation patterns are discussed.
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PMID:Energy conservation by succinate decarboxylation in Veillonella parvula. 164 32

The anaplerotic hypothesis for insulin release postulates that an increased generation of malonyl-CoA, acyl residues and diacylglycerol in nutrient-stimulated pancreatic islets may couple the catabolism of nutrient secretagogues to more distal events in the secretory sequence. In the light of this hypothesis, pyruvate carboxylase activity was measured in rat pancreatic islets using two distinct radioisotopic procedures. The first procedure is based on the conversion of oxalacetate generated from pyruvate to 14C-labelled citrate in the presence of [1-14C]acetyl-CoA and citrate synthase. The second technique involves the conversion of 14C-labelled oxalacetate generated from [1-14C]pyruvate to radioactive aspartate in the presence of L-glutamate and glutamate-oxalacetate transaminase. Pyruvate carboxylase activity amounted to 10 pmol/min per islet, was restricted to mitochondria, displayed a Km for pyruvate close to 0.4 mM, and demonstrated dependency towards ATP (apparent Ka close to 0.1 mM), Mg2+ and acetyl-CoA. It is proposed that pyruvate carboxylase activity accounts for the generation of 14C-labelled amino acids other than alanine in islets exposed to D-[3,4-14C]glucose and participates to the pyruvate/citrate shuttle for the transport of acetyl-CoA out of the mitochondria in nutrient-stimulated islets.
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PMID:Hexose metabolism in pancreatic islets: pyruvate carboxylase activity. 176 3

A gene encoding pyruvate carboxylase has previously been isolated from Saccharomyces cerevisiae. We have isolated a second gene, PYC2, from the same organism also encoding a pyruvate carboxylase. The gene PYC2 is situated on the right arm of chromosome II between the DUR 1, 2 markers and the telomere. We localized the previously isolated gene, which we designate PYC1, to chromosome VII. Disruption of either of the genes did not produce marked changes in the phenotype. However, simultaneous disruption of both genes resulted in inability to grow on glucose as sole carbon source, unless aspartate was added to the medium. This indicates that in wild-type yeast there is no bypass for the reaction catalysed by pyruvate carboxylase. The coding regions of both genes exhibit a homology of 90% at the amino acid level and 85% at the nucleotide level. No appreciable homology was found in the corresponding flanking regions. No differences in the Km values for ATP or pyruvate were observed between the enzymes obtained from strains carrying inactive, disrupted versions of one or other of the genes.
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PMID:DNA sequences in chromosomes II and VII code for pyruvate carboxylase isoenzymes in Saccharomyces cerevisiae: analysis of pyruvate carboxylase-deficient strains. 192 79

A 42-year-old woman developed a flu-like illness and died 8 days later with Reye's syndrome (RS). There are 26 other cases of adult-onset RS reported. Biochemical, immunologic, and molecular studies of liver, brain, and skeletal muscle revealed a non-uniform decrease in several mitochondrial residual enzyme activities in liver and brain. Pyruvate carboxylase activity was negligible. Cross-reacting material was present in normal abundance in isolated liver mitochondria for several enzymes that had reduced catalytic activity including pyruvate carboxylase. Subunit II (encoded by mitochondrial DNA) and subunit IV (encoded by nuclear DNA) of cytochrome c oxidase also were present in normal abundance with normal electrophoretic mobility. These observations, combined with pertinent findings of other investigators, allow us to speculate that the intramitochondrial matrix chemical environment is disturbed by preceding pathophysiologic events resulting in a lowered ATP/ADP ratio. The lowered intramitochondrial energetic state interferes with the refolding and assembly of imported mitochondrial proteins, causing a loss of the catalytic efficiency of these enzymes. This explains the selective vulnerability of mitochondria in RS and the non-uniform, disproportionate loss of enzyme activity.
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PMID:Adult Reye's syndrome: a review with new evidence for a generalized defect in intramitochondrial enzyme processing. 194 14

In a reaction that is analogous to the phosphorylation of ADP from carboxyphosphate, pyruvate carboxylase catalyses the formation of ATP from carbamoyl phosphate and ADP at a rate that is about 0.3% of the pyruvate-carboxylation reaction and about 3% of the full reverse reaction. Acetyl-CoA stimulates the phosphorylation of ADP from carbamoyl phosphate but is not an essential requirement of the reaction. Mg2+ also stimulates the reaction, and in the range of Mg2+ concentrations considered the effect of V is much larger in the absence of acetyl-CoA than in its presence. Acetyl-CoA and Mg2+ may be acting in a co-operative way to stimulate the phosphorylation of ADP in a similar way to their effects on the pyruvate-carboxylation reaction. The phosphorylation of ADP by carbamoyl phosphate is also stimulated by the presence of biotin in the part of the active site where this reaction occurs, but again it is not absolutely required for the reaction to proceed. The pH profiles of the phosphorylation of ADP by carbamoyl phosphate indicate that there are at least two ionizable residues involved in the reaction, one of which probably has a role in the release of carbamate from the active site.
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PMID:Pyruvate carboxylase catalysis of phosphate transfer between carbamoyl phosphate and ADP. 199 Oct 40

Pi depletion of proximal tubule cells isolated from mouse kidney results in a decrease in the cell content of fructose-2,6-bisphosphate and an increase in the rate of gluconeogenesis from pyruvate, malate and succinate. Gluconeogenesis from glycerol is unaffected by Pi depletion. Introduction of fructose-2,6-bisphosphate into the cytosol of ATP-permeabilized cells is accompanied by a fall in gluconeogenesis. The presence of external Ca2+ stimulates gluconeogenesis. When cytosolic Ca2+ is raised to 1.8 microM by permeabilization, the resealed cells still require 2.5 mM Ca2+ in the bathing medium in order to perform gluconeogenesis at the maximum rate. Cells permeabilized in the presence of cAMP show a decreased rate of glucose production. Phorbol ester stimulates gluconeogenesis provided that the phorbol treatment is performed in the absence of Ca2+ ions. It is suggested that Pi depletion may stimulate pyruvate carboxylase activity and facilitate the entry of certain gluconeogenic substrates into mitochondria. It is also proposed that important aspects of the control of renal gluconeogenesis by parathyroid hormone are mediated by protein kinase C.
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PMID:Studies of the regulation of renal gluconeogenesis in normal and Pi depleted proximal tubule cells. 234 Jun 30


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