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)

We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.
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PMID:Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12. 636 74

The object of this work was to investigate the operation of enzymes of the citric acid cycle, the glyoxylate pathway, the glucose metabolism as well as of pyruvate and phosphoenolpyruvate carboxylases in Pseudomonas aeruginosa 640x capable of complete degradation of DDT under the conditions of cometabolism. The activity of isocitrate and glucose-6-phosphate dehydrogenases producing reduced NADP, which is required for reductive dechlorination of DDT, appears to be high. Pyruvate carboxylase and phosphoenolpyruvate carboxylase (EC 4.1.1.31.) function simultaneously in the culture. Differences in the pathways of anaplerotic carbon dioxide fixation were found in P. aeruginosa 640x and the collection strain of P. aeruginosa PAO incapable of DDT degradation.
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PMID:[Central metabolic characteristics of a Pseudomonas aeruginosa culture degrading DDT]. 680 12

Glucose and glutamine metabolism in several cultured mammalian cell lines (BHK, CHO, and hybridoma cell lines) were investigated by correlating specific utilization and formation rates with specific maximum activities of regulatory enzymes involved in glycolysis and glutaminolysis. Results were compared with data from two insect cell lines and primary liver cells. Flux distribution was measured in a representative mammalian (BHK) and an insect (Spodoptera frugiperda) cell line using radioactive substrates. A high degree of similarity in many aspects of glucose and glutamine metabolism was observed among the cultured mammalian cell lines examined. Specific glucose utilization rates were always close to specific hexokinase activities, indicating that formation of glucose-6-phosphate from glucose (catalyzed by hexokinase) is the rate limiting step of glycolysis. No activity of the key enzymes connecting glycolysis with the tricarboxylic acid cycle, such as pyruvate dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase, could be detected. Flux distribution in BHK cells showed glycolytic rates very similar to lactate formation rates. No glucose- or pyruvate-derived carbon entered the tricarboxylic acid cycle, indicating that glucose is mainly metabolized via glycolysis and lactate formation. About 8% of utilized glucose was metabolized via the pentose phosphate shunt, while 20 to 30% of utilized glucose followed pathways other than glycolysis, the tricarboxylic acid cycle, or the pentose phosphate shunt. About 18% of utilized glutamine was oxidized, consistent with the notion that glutamine is the major energy source for mammalian cell lines. Mammalian cells cultured in serum-free low-protein medium showed higher utilization rates, flux rates, and enzyme activities than the same cells cultured in serum-supplemented medium. Insect cells oxidized glucose and pyruvate in addition to glutamine. Furthermore, insect cells produced little or no lactate and were able to channel glycolytic intermediates into the tricarboxylic acid cycle. Metabolic profiles of the type presented here for a variety of cell lines may eventually enable one to interfere with the metabolic patterns of cells relevant to biotechnology, with the hope of improving growth rate and/or productivity.
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PMID:Comparative analysis of glucose and glutamine metabolism in transformed mammalian cell lines, insect and primary liver cells. 855 65

The effect of nonhydrolyzable guanine nucleotides on mammalian acetyl CoA carboxylase (ACC) activity was examined. Using porous rat adipocytes and crude fat cell homogenates to study metabolic pathway flux, GMPPNP and/or GTP gamma S inhibited [14C]fatty acid formation by up to 95% when either [6-14C]glucose-6-phosphate or [1-14C]acetyl CoA was used as substrate. If [2-14C]malonyl CoA initiated flux, however, no inhibition was apparent. These pathway flux studies suggested that ACC was the locus of inhibition, and that the mechanism might involve a disruption of guanine nucleotide hydrolysis by the nonhydrolyzable analogues. Using partially and avidin-sepharose-purified ACC preparations from rat fat, liver and mammary tissue, citrate-stimulated ACC activity was inhibited by 25-75% with 50 microM GTP gamma S. Related compounds and nucleotides had absent-to-minimal effects on ACC. ATP gamma S was inhibitory (10-30% at 5-15 microM), but always to a lesser degree than equimolar GTP gamma S. Filter binding assays with [alpha-32P]GTP or [35S]GTP gamma S were negative, but low-level GTPase activity was detected. Using photoaffinity labelling techniques, [alpha-32P]GTP was found to bind ACC and not pyruvate carboxylase. The hypothesis that citrate-responsive ACC activity may be modulated by an intrinsic or associated GTP binding site is explored. Since ACC forms polymers, as does the cytoskeletal protein beta-tubulin, amino acid sequence comparisons between ACC and atypical GTP binding domain of beta tubulin are presented.
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PMID:Inhibition of acetyl CoA carboxylase by GTP gamma S. 960 94

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.
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PMID:Glucose-fatty acid cycle to inhibit glucose utilization and oxidation is not operative in fatty acid-cultured islets. 1048 Jun 4

The gram-positive bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, e.g. of L-glutamate and L-lysine. During the last 15 years, genetic engineering and amplification of genes have become fascinating methods for studying metabolic pathways in greater detail and for the construction of strains with the desired genotypes. In order to obtain a better understanding of the central metabolism and to quantify the in vivo fluxes in C. glutamicum, the [13C]-labelling technique was combined with metabolite balancing to achieve a unifying comprehensive pathway analysis. These methods can determine the flux distribution at the branch point between glycolysis and the pentose phosphate pathway. The in vivo fluxes in the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified glucose-6-phosphate and 6-phosphogluconate dehydrogenases determined in vitro were in full accordance with the fluxes measured by the [13C]-labelling technique. These data indicate that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH/NADP concentrations and the specific activity of glucose-6-phosphate dehydrogenase. The carbon flux via the oxidative pentose phosphate pathway correlated with the NADPH demand for L-lysine synthesis. Although it has generally been accepted that phosphoenolpyruvate carboxylase fulfills a main anaplerotic function in C. glutamicum, we recently detected that a biotin-dependent pyruvate carboxylase exists as a further anaplerotic enzyme in this bacterium. In addition to the activities of these two carboxylases three enzymes catalysing the decarboxylation of the C4 metabolites oxaloacetate or malate are also present in this bacterium. The individual flux rates at this complex anaplerotic node were investigated by using [13C]-labelled substrates. The results indicate that both carboxylation and decarboxylation occur simultaneously in C. glutamicum so that a high cyclic flux of oxaloacetate via phosphoenolpyruvate to pyruvate was found. Furthermore, we detected that in C. glutamicum two biosynthetic pathways exist for the synthesis of DL-diaminopimelate and L-lysine. As shown by NMR spectroscopy the relative use of both pathways in vivo is dependent on the ammonium concentration in the culture medium. Mutants defective in one pathway are still able to synthesise enough L-lysine for growth, but the L-lysine yields with overproducers were reduced. The luxury of having these two pathways gives C. glutamicum an increased flexibility in response to changing environmental conditions and is also related to the essential need for DL-diaminopimelate as a building block for the synthesis of the murein sacculus.
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PMID:Pathway analysis and metabolic engineering in Corynebacterium glutamicum. 1107 21

1-Methyl-4-phenylpyridinium (MPP+) is a mitochondrial Complex I inhibitor and is frequently used to investigate the pathological degeneration of neurons associated with Parkinson's disease (PD). In vitro, extracellular concentration of glucose is one of the most critical factors in establishing the vulnerability of neurons to MPP+ toxicity. While glucose is the primary energy fuel for the brain, central nervous system (CNS) neurons can also take up and utilize other metabolic intermediates for energy. In this study, we compared various monosaccharides, disaccharides, nutritive/non-nutritive sugar alcohols, glycolytic and gluconeogenic metabolic intermediates for their cytoprotection against MPP+ in murine brain neuroblastoma cells. Several monosaccharides were effective against MMP+ (500 microM) including glucose, fructose and mannose, which restored cell viability to 109 +/- 5%, 70 +/- 5%, 99 +/- 3% of live controls, respectively. Slight protective effects were observed in the presence of 3-phosphoglyceric acid and glucose-6-phosphate; however, no protective effects were exhibited by galactose, sucrose, sorbitol, mannitol, glycerol or various gluconeogenic and ketogenic amino acids. On the other hand, fructose 1,6 bisphosphate and gluconeogenic energy intermediates [pyruvic acid, malic acid and phospho(enol)pyruvate (PEP)] were neuroprotective against MPP+. The gluconeogenic intermediates elevated intracellular levels of ATP and reduced propidium iodide (PI) nucleic acid staining to live controls, but did not alter the MPP(+)-induced loss of mitochondrial O2 consumption. These data indicate that malic acid, pyruvic acid and PEP contribute to anaerobic substrate level phosphorylation. The use of hydrazine sulfate to impede gluconeogenesis through PEP carboxykinase (PEPCK) inhibition heightened the protective effects of energy substrates possibly due to attenuated ATP demands from pyruvate carboxylase (PC) activity and pyruvate mitochondrial transport. It was concluded from these studies that several metabolic intermediates are effective in fueling anaerobic glycolysis during mitochondrial inhibition by MPP+.
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PMID:The role of glycolysis and gluconeogenesis in the cytoprotection of neuroblastoma cells against 1-methyl 4-phenylpyridinium ion toxicity. 1256 89

Metabolic flux analysis based on 13C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF- mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF- mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.
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PMID:Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations. 1280 31

Cellular energy metabolism correlates with cell fate, but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood. Using a previously established chES cell model and electron microscopy (EM), we found that undifferentiated chES cells stored glycogen. Additionally, undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells, suggesting that chES cells direct glucose flux towards the glycogenic pathway. Moreover, we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway, as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs. Additionally, cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining, but it could be rescued by exogenous G6P. However, we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining. Moreover, differentiated chES cells expressed higher levels of GLUT1, HK1 and PFK mRNAs, while the level of GYS mRNA remained similar in control CEF cells. These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P, while differentiated chES cells have a decreased glycogen reserve, which suggests that the amount of glycogen is indicative of the chES cell state.
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PMID:Metabolic properties of chicken embryonic stem cells. 2110 67

Lactate and ammonia are the most important waste products of central carbon metabolism in mammalian cell cultures. In particular during batch and fed-batch cultivations these toxic by-products are excreted into the medium in large amounts, and not only affect cell viability and productivity but often also prevent growth to high cell densities. The most promising approach to overcome such a metabolic imbalance is the replacement of one or several components in the culture medium. It has been previously shown that pyruvate can be substituted for glutamine in cultures of adherent Madin-Darby canine kidney (MDCK) cells. As a consequence, the cells not only released no ammonia but glucose consumption and lactate production were also reduced significantly. In this work, the impact of media changes on glucose and glutamine metabolism was further elucidated by using a high-throughput platform for enzyme activity measurements of mammalian cells. Adherent MDCK cells were grown to stationary and exponential phase in six-well plates in serum-containing GMEM supplemented with glutamine or pyruvate. A total number of 28 key metabolic enzyme activities of cell extracts were analyzed. The overall activity of the pentose phosphate pathway was up-regulated during exponential cell growth in pyruvate-containing medium suggesting that more glucose-6-phosphate was channeled into the oxidative branch. Furthermore, the anaplerotic enzymes pyruvate carboxylase and pyruvate dehydrogenase showed higher cell specific activities with pyruvate. An increase in cell specific activity was also found for NAD(+)-dependent isocitrate dehydrogenase, glutamate dehydrogenase, and glutamine synthetase in MDCK cells grown with pyruvate. It can be assumed that the increase in enzyme activities was required to compensate for the energy demand and to replenish the glutamine pool. On the other hand, the activities of glutaminolytic enzymes (e.g., alanine and aspartate transaminase) were decreased in cells grown with pyruvate, which seems to be related to a decreased glutamine metabolism.
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PMID:Metabolic adaptation of MDCK cells to different growth conditions: effects on catalytic activities of central metabolic enzymes. 2161 69


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