Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A method for the removal of CoASH from tissue extracts by maleic anhydride is described. It eliminates CoASH interference in the acetyl-CoA cycling assay using phosphotransacetylase and citrate synthase. Maleyl-CoA thioether does not hydrolyze under the conditions of the assay and allows a reduction in the number of blank samples during acetyl-CoA determination. The levels of acetyl-CoA in whole rat brain, isolated synaptosomes, and mitochondria were found to be 61, 8.6, and 31.3 pmol/mg of protein, respectively.
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PMID:Elimination of CoASH interference from acetyl-CoA cycling assay by maleic anhydride. 367 77

Maximum activities of some key enzymes of metabolism were studied in elicited (inflammatory) macrophages of the mouse and lymph-node lymphocytes of the rat. The activity of hexokinase in the macrophage is very high, as high as that in any other major tissue of the body, and higher than that of phosphorylase or 6-phosphofructokinase, suggesting that glucose is a more important fuel than glycogen and that the pentose phosphate pathway is also important in these cells. The latter suggestion is supported by the high activities of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. However, the rate of glucose utilization by 'resting' macrophages incubated in vitro is less than the 10% of the activity of 6-phosphofructokinase: this suggests that the rate of glycolysis is increased dramatically during phagocytosis or increased secretory activity. The macrophages possess higher activities of citrate synthase and oxoglutarate dehydrogenase than do lymphocytes, suggesting that the tricarboxylic acid cycle may be important in energy generation in these cells. The activity of 3-oxoacid CoA-transferase is higher in the macrophage, but that of 3-hydroxybutyrate dehydrogenase is very much lower than those in the lymphocytes. The activity of carnitine palmitoyltransferase is higher in macrophages, suggesting that fatty acids as well as acetoacetate could provide acetyl-CoA as substrate for the tricarboxylic acid cycle. No detectable rate of acetoacetate or 3-hydroxybutyrate utilization was observed during incubation of resting macrophages, but that of oleate was 1.0 nmol/h per mg of protein or about 2.2% of the activity of palmitoyltransferase. The activity of glutaminase is about 4-fold higher in macrophages than in lymphocytes, which suggests that the rate of glutamine utilization could be very high. The rate of utilization of glutamine by resting incubated macrophages was similar to that reported for rat lymphocytes, but was considerably lower than the activity of glutaminase.
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PMID:Metabolism of glucose, glutamine, long-chain fatty acids and ketone bodies by murine macrophages. 380 Sep 71

The non-Michaelis-Menten kinetics, burst and steady-state periods, expressed by citrate synthase in the presence of citryl-CoA, were investigated by labelling experiments with trace amounts of [14C]acetyl-CoA. The results indicate that citrate becomes labelled in the reaction of liberated acetyl-CoA with the binary synthase.oxaloacetate complex that is transiently generated in the lyase reaction of citryl-CoA. Mediated by the hydrolase function of synthase, the counteracting citryl-CoA lyase and ligase reactions operate towards a transient flow equilibrium. This precedes the thermodynamic equilibrium and is established during the burst period; it is maintained under steady-state conditions and corresponds to the formation of transiently nonproductive synthase. The rates of both synthase partial reactions, therefore, are likewise affected. Oxaloacetate in the presence of acetyl-CoA competitively inhibits the hydrolysis of citryl-CoA and vice versa. In the synthase dependence of the burst periods and during the time dependence of the steady-state periods, nonproportionally more of physiological substrates participate in citrate formation. The nonproportional increase is a consequence of the continuously changing conditions to establish or to maintain the flow equilibrium, respectively, during the reaction progress. Third rate periods after the steady state result if the equilibrium conditions cannot be satisfied. High concentrations of oxaloacetate inhibit the expression of non-Michaelis-Menten kinetics by formation of nonproductive synthase.oxaloacetate complex. The supply of acetyl-CoA is then sufficient and the formation of the flow equilibrium prevented. The implication of the results with structural work is discussed.
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PMID:Hysteretic behaviour of citrate synthase. The reaction mechanism and the exclusion of synthase being a hysteretic enzyme. 383 Jan 75

The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation.
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PMID:Evidence from 13C NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase. 397 85

A new micromethod for determination of acetyl-CoA and malonyl-CoA using malonate decarboxylase is described. This enzyme catalyzes decarboxylation of malonate in a cyclic manner and produces acetate in proportion to the amount of a given acyl-CoA, such as acetyl-CoA, malonyl-CoA or propionyl-CoA. The acetate generated is converted to acetylphosphate by acetate kinase (EC 2.7.2.1) added at the same time and is determined spectrophotometrically as acetohydroxamate. The sensitivity of this method is high enough to detect 10(-12) mol of acetyl-CoA or malonyl-CoA. The simplicity of the method allows more than 30 samples to be analyzed at the same time without any prior extraction step. Although this method does not distinguish between acetyl-CoA and malonyl-CoA, malonyl-CoA alone can be measured by elimination of acetyl-CoA with citrate synthetase (EC 4.1.3.7).
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PMID:Malonyl-CoA: acetyl-CoA cycling. A new micromethod for determination of acyl-CoAs with malonate decarboxylase. 397 11

The function of glycerophosphate and malate-aspartate shuttles during glucose metabolism in two strains of Ehrlich ascites tumor cells was evaluated by several experimental approaches. The activities of the enzymes involved in these shuttle systems were assayed in the cytosolic and mitochondrial compartments after cell fractionation by the digitonin method. The glycerophosphate shuttle can be ruled out because of the lack of relevant enzymatic activities, and the failure of glucose to increase rotenone-inhibited respiration. Analysis of glycolytic flux in the presence of aminooxyacetate indicates that the activity of malate-aspartate shuttle may be very low. Balance studies of glucose uptake and lactate production suggest the existence of other pathways for the reoxidation of cytosolic NADH, which are acetyl-CoA dependent. Estimation of citrate synthase and ATP citrate lyase, in addition to the observed high activity of malate dehydrogenase, suggests a malate-citrate shuttle.
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PMID:The function of redox shuttles during aerobic glycolysis in two strains of Ehrlich ascites tumor cells. 400 10

Citrate specimens derived from chiral acetates were converted to the CoA derivatives. These were reconverted with citrate synthase to citrates under conditions of either predominating hydrolytic burst or predominating steady-state period. The stereochemical purity of substrates and products was determined. Reversal of the synthase condensation step occurs under both conditions but is markedly increased during the steady-state period. The results indicate that citryl-CoA-derived acetyl-CoA and oxaloacetate create the steady-state conditions. The hydrolase state and the ligase/lyase state of the synthase predominate under burst and steady-state conditions, respectively. This result indicates a conversion of the hydrolase state into the ligase/lyase state during the transition.
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PMID:Hysteretic behaviour of citrate synthase. Isotopically chiral citryl-CoA reveals increased number of reversals of the synthase condensation step under steady-state conditions. 401 81

1. The effects of 2-oxo-4-methylpentanoate, 2-oxo-3-methylbutanoate and 2-oxo-3-methylpentanoate on the activity of pyruvate dehydrogenase (EC 1.2.4.1), citrate synthase (EC 4.1.3.7), acetyl-CoA carboxylase, (EC 6.4.1.2) and fatty acid synthetase derived from the brains of 14-day-old rats were investigated. 2. The pyruvate dehydrogenase enzyme activity was competitively inhibited by 2-oxo-3-methylbutanoate with respect to pyruvate with a K(i) of 2.04mm but was unaffected by 2-oxo-4-methylpentanoate or 2-oxo-3-methylpentanoate. 3. The citrate synthase activity was inhibited competitively (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~7.2mm) and 2-oxo-3-methylbutanoate (K(i)~14.9mm) but not by 2-oxo-3-methylpentanoate. 4. The acetyl-CoA carboxylase activity was not inhibited significantly by any of the 2-oxo acids investigated. 5. The fatty acid synthetase activity was competitively inhibited (with respect to acetyl-CoA) by 2-oxo-4-methylpentanoate (K(i)~930mum) and 2-oxo-3-methylpentanoate (K(i)~3.45mm) but not by 2-oxo-3-methylbutanoate. 6. Preliminary experiments indicate that 2-oxo-4-methylpentanoate and 2-oxo-3-phenylpropionate (phenylpyruvate) significantly inhibit the ability of intact brain mitochondria from 14-day-old rats to oxidize pyruvate. 7. The results are discussed with reference to phenylketonuria and maple-syrup-urine disease. A biochemical mechanism is proposed to explain the characteristics of these diseases.
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PMID:Differential effects of 2-oxo acids on pyruvate utilization and fatty acid synthesis in rat brain. 415 48

The necessity for adding an internal standard to liver extracts during fluorimetric estimation of acetyl-CoA by the malic dehydrogenase-citrate synthase reaction is demonstrated. Addition of acetyl-CoA completely compensates for the inhibitory action of some tissue components. Values for hepatic acetyl-CoA in fed and fasted rats are given.
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PMID:Internal standards in the estimation of acetyl-CoA in liver extracts. 429 62

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
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PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82


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