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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 protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.
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PMID:Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation. 1 68

Closed aorta working hearts perfused with 1 mM pyruvate were subjected to a 4-fold increase in work load by raising the left atrial filling pressure. Citric acid cycle flux, pyruvate uptake, and oxygen consumption rose 3-fold when cardiac output was increased. In the first 40 sec after the transition tissue glutamate and citrate fell by 22 and 45%, respectively, and there were reciprocal decreases in malate and aspartate. The ratio of creatine phosphate/creatine declined by 50% within 30 sec, with a corresponding increase in inorganic phosphate, but the fall in the ATP/ADP ratio was only 10%. During the first 10 sec the surface fluorescence from cardiac pyridine nucleotides fell by 30% and this change was synchronous with a sharp decline in the calculated adenine nucleotide phosphate potential. This suggests that heart mitochondrial respiration is controlled by the cytosolic phosphate potential, and that a state 4 to state 3 transition occurs when cardiac output is increased. Apparent disequilbrium of creatine phosphokinase can be explained by the compartmentation of most of the cardiac ADP within the mitochondria. Citric acid cycle flux was coordinated by activational interactions at citrate synthase, isocitrate dehydrogenase, and alpha-ketoglutarate dehydrogenase, but a transient imbalance between the individual cycle steps leads to a sharp peak of lactate production shortly after the work transition.
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PMID:Regulation of myocardial energy metabolism. 17 15

1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...
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PMID:The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation. 18 46

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.
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PMID:Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes. 20 32

Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.
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PMID:Purification and some properties of the citrate synthase from a marine Pseudomonas. 20 30

Certain key enzymes of alternative pathways of glucose metabolism, of amino acid metabolism and of redox systems have been measured in hydra and this profile compared with mammalian differentiated tissues with a view to locating pathways of specific importance in hydra. There was a marked constant proportionality in the major part of the enzymes investigated, the profile suggested a metabolic pattern geared to utilization of amino acids as a carbon source for biosynthesis and energy production and to the production and conservation of pyruvate. The importance of conversion to ionized forms was noted. The most notable specific proportion changes were the exceptionally low lactate dehydrogenase, malic enzyme and the relatively high citrate synthase. The proximal-distal gradients in hydra were examined and these gradients suggested a switch to a more anaerobic type of metabolism and an elevation of the pentose phosphate pathway as the basal region was approached. Measurements of the formation of 14CO2 from specifically labelled glucose provided additional evidence for the functional activity and polarity of the pentose phosphate pathway in hydra. The effect of oligomycin, which can reverse polarity in hydra, had a significant effect on gradients of enzymes eliminating all except that observed for G6P dehydrogenase. The profile suggested a movement towards a more anaerobic type of metabolism, in keeping with the known biochemical action of this inhibitor. It is suggested that redox states and/or phosphorylation states may be featured in the positional information of cells in hydra.
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PMID:Positional information and pattern regulation in hydra: enzyme profiles. 24 Sep 2

1. The contents of some intermediates of glycolysis, the citric acid cycle and adenine nucleotides have been measured in the freeze-clamped locust flight muscle at rest and after 10s and 3min flight. The contents of glucose 6-phosphate, pyruvate, alanine and especially fructose bisphosphate and triose phosphates increased markedly upon flight. The content of acetyl-CoA is decreased after 3min flight whereas that of acetylcarnitine is decreased markedly after 10s flight, but returns towards the resting value after 3min flight. The content of citrate is markedly decreased after both 10s and 3min flight, whereas that of isocitrate is changed very little after 10s and is increased by 50% after 3min. The content of oxaloacetate is very low in insect flight muscle and hence it was measured by a sensitive radiochemical assay. The content of oxaloacetate increased about 2-fold after 3min flight. A similar change was observed in the content of malate. The content of ATP decreased about 15%, whereas those of ADP and AMP increased about 2-fold after 3min flight. 2. Calculations based on O(2) uptake of the intact insect indicate that the rate of the citric acid cycle must be increased >100-fold during flight. Consequently, if citrate synthase catalyses a non-equilibrium reaction, the activity of the enzyme must increase >100-fold during flight. However, changes in the concentrations of possible regulators of citrate synthase, oxaloacetate, acetyl-CoA and citrate (which is an allosteric inhibitor), are not sufficient to account for this change in activity. It is concluded that there may be much larger changes in the free concentration of oxaloacetate than are indicated by the changes in the total content of this metabolite or that other unknown factors must play an additional role in the regulation of citrate synthase activity. 3. The increased content of oxaloacetate could be produced via pyruvate carboxylase, which may be stimulated during the early stages of flight by the increased concentration of pyruvate. 4. The decreases in the concentrations of citrate and alpha-oxoglutarate indicate that isocitrate dehydrogenase and oxoglutarate dehydrogenase may be stimulated by factors other than their pathway substrates during the early stages of flight. 5. Calculated mitochondrial and cytosolic NAD(+)/NADH ratios are both increased upon flight. The change in the mitochondrial ratio indicates the importance of the intramitochondrial ATP/ADP concentration ratio in the regulation of the rate of electron transfer in this muscle.
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PMID:Changes in the contents of adenine nucleotides and intermediates of glycolysis and the citric acid cycle in flight muscle of the locust upon flight and their relationship to the control of the cycle. 43 78

The activity of enzymes involved in carbon metabolism of Candida boidinii KDI was compared on media containing methanol with bicarbonate and without it. The presence of carbon dioxide stimulated the activity of methanol oxidase, formaldehyde dehydrogenase, hexulose phosphate synthase and particularly carboxylases of pyruvate and phosphoenol pyruvate. At the same time, the activity of formate dehydrogenase, citrate synthase and isocitrate lyase decreased. Therefore, carbon dioxide produces a significant effect on methylotrophic metabolism of Candida boidinii KDI and is actively involved in biosynthetic processes.
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PMID:[Effect of carbon dioxide on the methylotrophic metabolism of Candida boidinii]. 71 85

1. Enzyme activities (units/g wet wt.) were determined in the caput and cauda epididymidis and in epididymal spermatozoa of the rat. 2. The activity of most enzymes in the cauda was between 50 and 100% of that in the caput, except that ATP citrate lyase was barely detectable in the cauda. 3. Spermatozoa, unlike epididymal tissue, contained sorbitol dehydrogenase but lacked ATP citrate lyase. NADP+-malate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, succinate dehydrogenase, carnitine acetyltransferase and citrate synthase were 5 to 400 times as active in spermatozoa as in epididymal tissue. 4. 2-Oxoglutarate dehydrogenase was the least active member of the tricarboxylic acid cycle in all tissues and most closely matched the measured flux through the cycle. 5. The concentrations of hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase were equivalent to the more active enzymes of the tricarboxylic acid cycle, indicating the capacity for extensive lipid oxidation, and the presence of 3-hydroxybutyrate dehydrogenase suggests that these tissues can also oxidize ketone bodies. 6. Transfer of reducing equivalents from cytoplasm to mitochondrion is unlikely to occur by means of the glycerol phosphate cycle because mitochondrial glycerol 3-phosphate dehydrogenase is relatively inactive in epididymal tissue, whereas the cytoplasmic enzyme has little activity in spermatozoa, but transfer may be accomplished by the malate-aspartate shuttle. 7. Transfer of acetyl units from mitochondrion to cytoplasm could be effected by the pyruvate-malate cycle in the caput of androgen-maintained rats, but not in the other tissues because of the low activity of ATP citrate lyase. Acetyl unit transfer could take place via acetylcarnitine, mediated by carnitine acetyltransferase. 8. Castration resulted in a decrease in the concentration of nearly all enzymes, although subsequent administration of testosterone restored concentrations to values similar to those in animals maintained by endogenous androgen. The extent to which enzyme concentration was changed by an alteration in androgen status was highly variable, but was most marked in the case of pyruvate carboxylase.
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PMID:Activity and androgenic control of enzymes associated with the tricarboxylic acid cycle, lipid oxidation and mitochondrial shuttles in the epididymis and epididymal spermatozoa of the rat. 72 83

The pathway of acetate assimilation in Methanosarcina barkeri was determined from analysis of the position of label in alanine, aspartate, and glutamate formed in cells grown in the presence of [14C]acetate and by measurement of enzyme activities in cell extracts. The specific radioactivity of glutamate from cells grown on [1-14C]- or [2-14C]acetate was approximately twice that of aspartate. The methyl and carboxyl carbons of acetate were incorporated into aspartate and glutamate to similar extents. Degradation studies revealed that acetate was not significantly incorporated into the C1 of alanine, C1 or C4 of aspartate, or C1 of glutamate. The C5 of glutamate, however, was partially derived from the carboxyl carbon of acetate. Cell extracts were found to contain the following enzyme activities, in nanomoles per minute per milligram of protein at 37 degrees C: F420-linked pyruvate synthase, 170; citrate synthase, 0.7; aconitase, 55; oxidized nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, 75; and oxidized nicotinamide adenine dinucleotide-linked malate dehydrogenase, 76. The results indicate that M. barkeri assimilates acetate into alanine and aspartate via pyruvate and oxaloacetate and into glutamate via citrate, isocitrate, and alpha-ketoglutarate. The data reveal differences in the metabolism of M. barkeri and Methanobacterium thermoautotrophicum and similarities in the assimilation of acetate between M. barkeri and other anaerobic bacteria, such as Clostridium kluyveri.
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PMID:Acetate assimilation pathway of Methanosarcina barkeri. 76 16


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