Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

At 37 degrees C, pH 7.4, carbonic anhydrase activity (kenz) of disrupted rat renal proximal tubules and cortical mitochondria was 2.5 +/- 0.8 (n = 3) and 0.15 +/- 0.40 (n = 3) ml.mg-1.s-1, respectively. Turnover number for renal mitochondrial carbonic anhydrase (CA V) was 24,000 s-1. CA V activity of intact mitochondria was completely inhibited by 0.15 microM ethoxzolamide (EZ). Intact proximal tubules, prepared from 48-h starved male rats, were incubated at 37 degrees C in 10 mM pyruvate in Krebs-Henseleit bicarbonate saline buffer, 5% CO2-95% O2. The rate of glucose synthesis over 60 min was reduced 50% by including 0.6 microM EZ in the incubation solution. The concentration of NaHCO3 was doubled to 50 mM (with a corresponding decrease in NaCl) and the solution gassed with 10% CO2-90% O2; 2.4 microM EZ no longer decreased glucose synthesis. It was concluded that inhibition of glucose synthesis by EZ was directly a result of inhibiting the carbonic anhydrases. The rate of glucose production was subsequently determined with tubules incubating in a HCO3(-)-free N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (HEPES) buffer; this rate was decreased 50% by 0.6 microM EZ. These data support the hypotheses that CA V provides HCO3- for pyruvate carboxylase and that CO2 can be provided by tubular metabolism. Intact tubules were incubated in from 5 to 20 mM pyruvate in either 25 or 50 mM HCO3-; in either buffer, the rate of glucose synthesis was similar, increasing with increasing pyruvate concentration. At no pyruvate concentration was there a change in the rate of glucose production when tubules were incubated in 50 mM HCO3- buffer with 1.6 microM EZ. These data also support the hypothesis that CA V provides the HCO3- substrate for pyruvate carboxylation when there is a high rate of intracellular CO2 production and external CO2 is low. It is further concluded that the cytosolic carbonic anhydrase (CA II) and the membrane-bound carbonic anhydrase (CA IV) are not involved in glucose synthesis from pyruvate.
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PMID:Mitochondrial carbonic anhydrase is involved in rat renal glucose synthesis. 251 97

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.
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PMID:Inhibition of CA V decreases glucose synthesis from pyruvate. 309 76

Carbonic anhydrase V (CA V) is expressed in mitochondrial matrix in liver and several other tissues. It is of interest for its putative roles in providing bicarbonate to carbamoyl phosphate synthetase for ureagenesis and to pyruvate carboxylase for gluconeogenesis and its possible importance in explaining certain inherited metabolic disorders with hyperammonemia and hypoglycemia. Following the recent characterization of the cDNA for human CA V, we report the isolation of the human gene from two lambda genomic libraries and its characterization. The CA V gene (CA5) is approximately 50 kb long and contains 7 exons and 6 introns. The exon-intron boundaries are found in positions identical to those determined for the previously described CA II, CA III, and CA VII genes. Like the CA VII gene, CA5 does not contain typical TATA and CAAT promoter elements in the 5' flanking region but does contain a TTTAA sequence 147 nucleotides upstream of the initiation codon. CA5 also contains a 12-bp GT-rich segment beginning 13 bp downstream of the polyadenylation signal in the 3' untranslated region of exon 7. FISH analysis allowed CA5 to be assigned to chromosome 16q24.3. An unprocessed pseudogene containing sequence homologous to exons 3-7 and introns 3-6 was also isolated and was assigned by FISH analysis to chromosome 16p11.2-p12.
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PMID:Genomic organization of the human gene (CA5) and pseudogene for mitochondrial carbonic anhydrase V and their localization to chromosomes 16q and 16p. 749 83

The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.
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PMID:Differentiation-dependent expression of CA V and the role of carbonic anhydrase isozymes in pyruvate carboxylation in adipocytes. 864 47

In peripheral tissues, carbonic anhydrase (CA) inhibition secondarily decreases the anaplerotic activity of pyruvate carboxylase activity leading to a decline in citric acid cycle intermediates and glutamate. In view of the important role of pyruvate carboxylase in the brain, we examined the effects of CA inhibition on pyruvate-carboxylase-mediated [14C]CO2 fixation in cultured astrocytes from postnatal rat brains. Incubation with H[14C]O3 led to radiolabeling of metabolites found both in the cells and in the medium. These were separated by ion exchange chromatography for identification. Ethoxyzolamide (ETZ), a sulfonamide CA inhibitor (SCAI) with a heterocyclic side group, caused a 43-73% decrease in cell lysate [alpha-ketoglutarate] and 14C incorporation into major products of pyruvate carboxylation in the cell lysates and cell medium (i.e., released products). Half-maximal inhibition of [14C]CO2 fixation was observed between 1 and 3 x 10(7) M. This is similar to the IC50 value for ETZ inhibition of events in other cells that are thought to be mediated by CA. Inhibition was also observed with trifluormethanesulfonamide, an aliphatic SCAI, providing further evidence that this effect is mediated by CA. Western blot analysis using isozyme-specific antisera indicated that astrocytes contain CA II, a cytosolic isozyme, but CA III, CA IV and CA V could not be detected. This finding is unusual since the effects of SCAIs on pyruvate carboxylation in other tissues have been attributed to inhibition of the intramitochondrial isozyme. CA V. [14C]CO2 fixation was also decreased by lowering media [pyruvate] or by addition of 5 mM acetoacetate. It is hypothesized that SCAIs may inhibit pyruvate carboxylation in astrocytes by limiting the supply of bicarbonate to this enzyme while ketone bodies, by inhibiting glucose oxidation, may limit the supply of pyruvate. Interestingly, both SCAIs and ketogenic diets are used to treat adolescent forms of epilepsy. The possibility that these treatments might ultimately work by affecting anaplerotic pyruvate carboxylase activity in the brain is discussed.
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PMID:Effect of carbonic anhydrase inhibition and acetoacetate on anaplerotic pyruvate carboxylase activity in cultured rat astrocytes. 909 31

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3- to pyruvate carboxylase for the first step in gluconeogenesis and HCO3- to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I. (J Histochem Cytochem 47:517-524, 1999)
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PMID:Cell-specific expression of mitochondrial carbonic anhydrase in the human and rat gastrointestinal tract. 1008 53

Carbonic anhydrase (CA) V is a mitochondrial enzyme that has been reported in several tissues of the gastrointestinal tract. In liver, it participates in ureagenesis and gluconeogenesis by providing bicarbonate ions for two other mitochondrial enzymes: carbamyl phosphate synthetase I and pyruvate carboxylase. This study presents evidence of immunohistochemical localization of CA V in the rodent nervous tissue. Polyclonal rabbit antisera against a polypeptide of 17 C-terminal amino acids of rat CA V and against purified recombinant mouse isozyme were used in western blotting and immunoperoxidase stainings. Immunohistochemistry showed that CA V is expressed in astrocytes and neurons but not in oligodendrocytes, which are rich in CA II, or capillary endothelial cells, which express CA IV on their plasma face. The specificity of the immunohistochemical results was confirmed by western blotting, which identified a major 30-kDa polypeptide band of CA V in mouse cerebral cortex, hippocampus, cerebellum, spinal cord, and sciatic nerve. The expression of CA V in astrocytes and neurons suggests that this isozyme has a cell-specific, physiological role in the nervous system. In astrocytes, CA V may play an important role in gluconeogenesis by providing bicarbonate ions for the pyruvate carboxylase. The neuronal CA V could be involved in the regulation of the intramitochondrial calcium level, thus contributing to the stability of the intracellular calcium concentration. CA V may also participate in bicarbonate ion-induced GABA responses by regulating the bicarbonate homeostasis in neurons, and its inhibition could be the basis of some neurotropic effects of carbonic anhydrase inhibitors.
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PMID:Mitochondrial carbonic anhydrase in the nervous system: expression in neuronal and glial cells. 1103 10

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.
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PMID:Mitochondrial carbonic anhydrase VA deficiency resulting from CA5A alterations presents with hyperammonemia in early childhood. 2453 Feb 3

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