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)

Pancreatic islets were cultured for 24 h in the presence of 1 mM glucose, which renders islets incapable of responding to glucose with insulin release. These islets were compared to islets maintained at 20 mM glucose for 24 h. Detritiation of [2-3H]glucose and [5-3H]glucose in 1 mM glucose islets was normal, suggesting that glucose transport and phosphorylation and all enzymes of glycolysis were not down-regulated in the incapacitated islets. 14CO2 formation from [U-14C]glucose and [6-14C]glucose was inhibited up to 80% and 14CO2 from methyl succinate was inhibited up to 60%, indicating that down-regulation at (a) mitochondrial site(s) might explain the incapacitated insulin release. 14CO2 formation from [3,4-14C]glucose (which becomes [1-14C]pyruvate) was decreased, indicating that the reaction catalyzed by pyruvate dehydrogenase was down-regulated. This decrease, however, was not as large as the decreases in 14CO2 formation from [U-14C]glucose, [2-14C]glucose (which becomes [2-14C]pyruvate), or [6-14C]glucose (which becomes [3-14C]pyruvate), indicating that other reactions were also down-regulated. 14CO2 formation from [1-14C]glucose was inhibited less than that from [6-14C]glucose in the incapacitated islets (34 vs 54%) and these rates indicated that flux of glucose through the pentose phosphate pathway was increased in the incapacitated islet, such that 29% (0.4 nmol of 1.4 glucose/100 islets/90 min) was metabolized via this pathway in the incapacitated islet but only 3.4% (0.1 of 2.9 nmol glucose/100 islets/90 min) was metabolized via the pentose pathway in the 20 mM glucose islets. With rates of 14CO2 evolved from glucose labeled at C2 and C6 and from methyl succinate labeled at C1 + C4 and C2 + C3 the 14CO2 ratio formula was used to calculate the ratios of carboxylated and decarboxylated pyruvate. Roughly equal amounts of pyruvate entered the citric acid cycle by each route in islets maintained for 24 h at 1, 5, or 20 mM glucose. The results indicate that decarboxylation and carboxylation of pyruvate were about equally suppressed in incapacitated islets and that direct inhibition of reactions of the cycle was unlikely. This is consistent with evidence which indicates that down-regulation of both pyruvate carboxylase and pyruvate dehydrogenase occurs in incapacitated islets, i.e., under long-term conditions that modify amounts of enzymes (MacDonald et al., 1991, J. Biol. Chem. 266, 22392-22397).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Estimates of glycolysis, pyruvate (de)carboxylation, pentose phosphate pathway, and methyl succinate metabolism in incapacitated pancreatic islets. 837 57

Gluconeogenesis from isotopically substituted (3-13C)alanine (Ala) was demonstrated in the last larval instar of an insect, Manduca sexta, when maintained on low carbohydrate diets. 13C was incorporated into all carbons of the blood sugar trehalose (Tre), but enrichments of C1 and C6, and C2 and C5 were greatest. Relative to the amount of [3-13C]Ala metabolized, larvae maintained on a low carbohydrate diet supplemented with casein displayed the greatest enrichment of Tre. Very little de novo synthesis of Tre was observed in larvae maintained on a complete-balanced diet containing calorically equivalent amounts of sucrose and casein. Starvation failed to induce gluconeogenesis and 13C was not incorporated into Tre in starved insects. Activity of the TCA cycle contributed approximately 10% of the 13C incorporated into Tre in larvae on low carbohydrate diets, while the TCA cycle contribution in larvae on the complete diet approached 70%. The pattern of 13C enrichment of glucose in larvae on the low carbohydrate diets indicated that cytoplasmic carboxylation, possibly due to 'malic enzyme'-like activity, contributed significantly to the synthesis of Tre. The pentose phosphate pathway was evidenced in insects on all diets. Glucose labelling ratios indicated a pentose cycling flux of 10 to 20% in insects on the low carbohydrate diets and 50% in larvae on the complete diet. Glutamine together with lesser amounts of glutamate and glutathione were also products of the labelled Ala. The distribution of label in these products under different dietary conditions demonstrated shifts in the relative contribution of pyruvate carboxylase and pyruvate dehydrogenase activities for providing substrate to the TCA cycle. In the expected fashion starved insects and insects on the low carbohydrate diets incorporated a greater proportion of 13C into the TCA cycle via carboxylation while incorporation by the two pathways was similar in insects on the complete diet. The significance of these findings with regard to the regulation of gluconeogenesis in M. sexta and comparison of the present results with those obtained from studies of hepatic gluconeogenesis are discussed.
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PMID:Gluconeogenesis and effect of nutritional status on TCA cycle activity in the insect Manduca sexta. 854 15

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

Previous studies in rat islets have suggested that anaplerosis plays an important role in the regulation of pancreatic beta cell function and growth. However, the relative contribution of islet beta cells versus non-beta cells to glucose-regulated anaplerosis is not known. Furthermore, the fate of glucose carbon entering the Krebs cycle of islet cells remains to be determined. The present study has examined the anaplerosis of glucose carbon in purified rat beta cells using specific 14C-labeled glucose tracers. Between 5 and 20 mM glucose, the oxidative production of CO2 from [3,4-14C]glucose represented close to 100% of the total glucose utilization by the cells. Anaplerosis, quantified as the difference between 14CO2 production from [3,4-14C]glucose and [6-14C]glucose, was strongly influenced by glucose, particularly between 5 and 10 mM. The dose dependence of glucose-induced insulin secretion correlated with the accumulation of citrate and malate in beta(INS-1) cells. All glucose carbon that was not oxidized to CO2 was recovered from the cells after extraction in trichloroacetic acid. This indirectly indicates that lactate output is minimal in beta cells. From the effect of cycloheximide upon the incorporation of 14C-glucose into the acid-precipitable fraction, it could be calculated that 25% of glucose carbon entering the Krebs cycle via anaplerosis is channeled into protein synthesis. In contrast, non-beta cells (approximately 80% glucagon-producing alpha cells) exhibited rates of glucose oxidation that were (1)/(3) to (1)/(6) those of the total glucose utilization and no detectable anaplerosis from glucose carbon. This difference between the two cell types was associated with a 7-fold higher expression of the anaplerotic enzyme pyruvate carboxylase in beta cells, as well as a 4-fold lower ratio of lactate dehydrogenase to FAD-linked glycerol phosphate dehydrogenase in beta cells versus alpha cells. Finally, glucose caused a dose-dependent suppression of the activity of the pentose phosphate pathway in beta cells. In conclusion, rat beta cells metabolize glucose essentially via aerobic glycolysis, whereas glycolysis in alpha cells is largely anaerobic. The results support the view that anaplerosis is an essential pathway implicated in beta cell activation by glucose.
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PMID:Metabolic fate of glucose in purified islet cells. Glucose-regulated anaplerosis in beta cells. 922 23

The metabolism of glucose is deranged in thiamin deficiency, but once any deficiency has been corrected there is no further effect of increased thiamin intake on the ability to metabolize glucose through either pyruvate dehydrogenase (EC 1.2.4.1) and the citric acid cycle, or the pentose phosphate pathway, in which transketolase (EC 2.2.1.1) is the thiamin-dependent step. It has been suggested that the Wernicke-Korsakoff syndrome is associated with a genetic variant of transketolase which requires a higher than normal concentration of thiamin diphosphate for activity. This finding would suggest that there may be a group of the population who have a higher than average requirement for thiamin, but the evidence is not convincing. There are no estimates of biotin requirements, but either coenzyme saturation of erythrocyte pyruvate carboxylase, or the excretion of 3-hydroxy-isovalerate (perhaps after a test dose of leucine) could be used to assess requirements in depletion-repletion studies. Biotin deficiency leads to impaired glucose tolerance, but it is unlikely that glucose tolerance could be used to assess optimum biotin status, since other more common factors affect glucose tolerance to a greater extent. Plasma triacylglycerol and nonesterified fatty acids are moderately elevated in pantothenic acid deficiency. However, this is unlikely to be useful in assessing pantothenate status, since again, other more common factors affect plasma lipids. To date there are no biochemical indices of adequate pantothenate nutrition, and no estimates of requirements.
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PMID:Optimum nutrition: thiamin, biotin and pantothenate. 1046 87

Alanine is the major amino acid utilized by the liver for gluconeogenesis under normal conditions. The metabolism of alanine in rat liver was investigated by means of (1)H and (13)C NMR spectroscopic studies in vivo and in vitro after infusion of L- and D-alanine labelled with (13)C at the carboxyl and methyl group into normal, fasted rats. Valuable information about different metabolic pathways of alanine in rat liver and their regulation under the conditions of gluconeogenesis were obtained. The enrichment of the alanine pool by the infusate was estimated to be 11% for L-alanine and 70% for D-alanine. After infusion of labelled D-alanines, the metabolic pathway via D-amino acid oxidase was observed. The labelled alanines entered the tricarboxylic acid cycle mainly via pyruvate carboxylase; the ratio of pyruvate dehydrogenase activity to that of pyruvate carboxylase is about 28%. The ratio of flux from phosphoenolpyruvate (PEP) through phosphoenolpyruvate kinase as compared with the flux from PEP to glucose was approximately 42%. From the labelling pattern of glucose it was concluded that the pentose phosphate cycle was active under the experimental conditions.
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PMID:Metabolism of D- and L-[(13)C]alanine in rat liver detected by (1)H and (13)C NMR spectroscopy in vivo and in vitro. 1079 35

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

The technique of metabolic flux analysis was implemented to elucidate the flux balancing of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment. The results showed that the majority of the substrate (97.70 +/- 0.49%) was funneled into the glycolytic pathway, while the remainder was subdivided between the pentose phosphate pathway and pathways for polysaccharide synthesis. At the pyruvate node, 87.30 +/- 1.38% of the flux was channeled through the reaction governed by pyruvate decarboxylase. Fluxes through the pyruvate dehydrogenase bypass were maintained at a constant level (82.65 +/- 1.47%) irrespective of the configuration of the fermentation setup. Activity through the TCA "cycle" was replenished by the reaction catalyzed by pyruvate carboxylase and by the transport of cytosolic oxaloacetate across the mitochondrial membrane. The CO(2) evolution rate varied as fermentation progressed; however, the yield coefficient of CO(2) remained at a constant value. Although a constant yield of ethanol (0.42 g of ethanol/g of glucose) was obtained, operations of the TCA cycle were gradually switched from partially reductive to partially oxidative pathways from the first fermenter to the fourth fermenter.
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PMID:Metabolic flux variation of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment. 1173 40

Nuclear magnetic resonance was used as the primary technique to investigate the effect of ethanol (40, 80, and 160 mM) on the levels of high-energy phosphates, glycolytic flux, anaplerotic and oxidative fluxes to the tricarboxylic acid (TCA) cycle, the contribution of the pentose phosphate pathway (PPP), and the uptake and release of amino acids on primary cultures of rat astrocytes. On line (31)P-NMR spectroscopy showed that long-term exposure to ethanol caused a drop in the levels of ATP and phosphocreatine. The ratio between the fluxes through the pyruvate dehydrogenase and pyruvate carboxylase reactions also decreased, whereas the glycolytic flux and the ratio between formation of lactate and glucose consumption increased when cells were exposed to acute doses of ethanol. Flux through the pentose phosphate pathway was not affected. The uptake of cysteine and the release of glutamine were stimulated by ethanol, whereas the release of methionine was inhibited. Moreover, the fractional enrichment in serine was enhanced. The changes in the amino acid metabolism are interpreted as a response to oxidative stress induced by ethanol.
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PMID:Effect of ethanol on the metabolism of primary astrocytes studied by (13)C- and (31)P-NMR spectroscopy. 1174 5

L-Lysine has been manufactured using Corynebacterium glutamicum for more than 40 years. Nowadays production exceeds 600,000 tons per year. Based on conventionally bred strains, further improvement of lysine productivity has been achieved by genetic engineering. Pyruvate carboxylase, aspartate kinase, dihydrodipicolinate synthase, homoserine dehydrogenase and the specific lysine exporter were shown to be key enzymes for lysine production and were characterized in detail. Their combined engineering led to a striking increase in lysine formation. Pathway modeling with data emerging from 13C-isotope experiments revealed a coordinated flux through pentose phosphate cycle and tricarboxylic acid cycle and intensive futile cycling between C3 compounds of glycolysis and C4 compounds of tricarboxylic acid cycle. Process economics have been optimized by developing repeated fed-batch techniques and technical continuous fermentations. In addition, on-line metabolic pathway analysis or flow cytometry may help to improve the fermentation performance. Finally, the availability of the Corynebacterium glutamicum genome sequence has a major impact on the improvement of the biotechnological manufacture of lysine. In this context, all genes of the carbon flow from sugar uptake to lysine secretion have been identified and are accessible to manipulation. The whole sequence information gives access to post genome technologies such as transcriptome analysis, investigation of the proteome and the active metabolic network. These multi-parallel working technologies will accelerate the generation of knowledge. For the first time there is a chance of understanding the overall picture of the physiological state of lysine overproduction in a technical environment.
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PMID:Biotechnological manufacture of lysine. 1252 89


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