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:2.3.3.1 (citrate synthase)
4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We isolated and characterized mutants defective in nuo, encoding NADH dehydrogenase I, the multisubunit complex homologous to eucaryotic mitochondrial complex I. By Southern hybridization and/or sequence analysis, we characterized three distinct mutations: a polar insertion designated nuoG::Tn10-1, a nonpolar insertion designated nuoF::Km-1, and a large deletion designated delta(nuoFGHIJKL)-1. Cells carrying any of these three mutations exhibited identical phenotypes. Each mutant exhibited reduced NADH oxidase activity, grew poorly on minimal salts medium containing acetate as the sole carbon source, and failed to produce the inner, L-aspartate chemotactic band on tryptone swarm plates. During exponential growth in tryptone broth, nuo mutants grew as rapidly as wild-type cells and excreted similar amounts of acetate into the medium. As they began the transition to stationary phase, in contrast to wild-type cells, the mutant cells abruptly slowed their growth and continued to excrete acetate. The growth defect was entirely suppressed by L-serine or D-pyruvate, partially suppressed by alpha-ketoglutarate or acetate, and not suppressed by L-aspartate or L-glutamate. We extended these studies, analyzing the sequential consumption of amino acids by both wild-type and nuo mutant cells growing in tryptone broth. During the lag and exponential phases, both wild-type and mutant cells consumed, in order, L-serine and L-aspartate. As they began the transition to stationary phase, both cell types consumed L-tryptophan. Whereas wild-type cells then consumed L-glutamate, glycine, L-threonine, and L-alanine, mutant cells utilized these amino acids poorly. We propose that cells defective for NADH dehydrogenase I exhibit all these phenotypes, because large NADH/NAD+ ratios inhibit certain tricarboxylic acid cycle enzymes, e.g., citrate synthase and malate dehydrogenase.
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PMID:Mutations in NADH:ubiquinone oxidoreductase of Escherichia coli affect growth on mixed amino acids. 815 82

The human leukaemic cell line HL60 undergoes differentiation to granulocyte-like cells in response to dimethylsulphoxide (DMSO). The rates of glucose and glutamine utilization were studied in HL60 cells that were either undifferentiated or fully differentiated by 9 days exposure to DMSO. Differentiation did not alter the rate of utilization of exogenous glucose, approximately 75% of which was converted to lactate in each case. The activities of hexokinase, phosphofructokinase, pyruvate kinase and citrate synthase were similarly unaffected. In contrast, the activity of the oxidative segment of the pentose-phosphate pathway was enhanced by differentiation, and no glycogen synthase activity could be detected. These observations are consistent with the significantly lower content of glycogen, the increased activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the increased oxidation of [1-14C] glucose relative to [6-14C] glucose in the differentiated cells. Glucose utilization was depressed by exogenous glutamine but, at the same time, glutamine utilization was enhanced by glucose in both cell types; these reciprocal effects were more pronounced in the undifferentiated HL60 cells. Glucose utilization may be depressed in the presence of glutamine as a result of the allosteric inhibition of a rate-limiting step of glycolysis (eg. phosphofructokinase). In spite of having glutaminase activity twice that of their differentiated counterparts, the uptake of glutamine by undifferentiated HL60 cells was low, especially when it was the sole substrate. The stimulation of glutaminolysis by glucose may be due to activation of mitochondrial glutamine transport. A large proportion of the glutamine utilized by both cells contributed to a net accumulation of glutamate, aspartate and alanine, whilst up to 35% was oxidized to CO2. In contrast, almost all of the glucose utilized was converted to lactate and very little was oxidized. The high rates of glycolysis and glutaminolysis observed before and after differentiation may not contribute primarily to energy production but may supply, in undifferentiated cells, substrates for biosynthetic processes that generate nucleic acid precursors or, in the case of differentiated cells which synthesize reactive oxygen intermediates, substrates that maintain NADP in a reduced state.
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PMID:Glycolytic, glutaminolytic and pentose-phosphate pathways in promyelocytic HL60 and DMSO-differentiated HL60 cells. 833 14

1. The metabolism of glucose, glutamine and ketone-bodies was studied in the small intestine of rats after 5 days of hyperthyroidism. 2. Portal-drained visceral bloodflow increased by 20.1% (P < 0.05) in hyperthyroid rats and was accompanied by a decrease in the arteriovenous concentration difference of glutamine (25.7%, P < 0.05), glutamate (22.0%, P < 0.05), alanine (20.9%, P < 0.05) and ammonia (20.6%, P < 0.05) and an increase in that of glucose (27.2%, P < 0.05), lactate (28.9%, P < 0.05) and ketone-bodies (163.2%, P < 0.001). 3. The gut of hyperthyroid rats showed increased rates of extraction of glucose, lactate and ketone-bodies. 4. Enterocytes isolated from hyperthyroid rats showed increased rates of utilization of glucose and ketone-bodies but that of glutamine were decreased. 5. The maximal activities of hexokinase, 6-phosphofructokinase, pyruvate kinase, citrate synthase and oxoglutarate dehydrogenase were increased (by 13.7-36.2%) in intestinal mucosal scrapings of hyperthyroid rats, whereas the activity of glutaminase was decreased (22.1-31.4%). 6. It is concluded that hyperthyroidism increases the rates of utilization of glucose and ketone-bodies but decreases that of glutamine (both in vivo and in vitro) by the epithelial cells of the small intestine.
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PMID:Effects of hyperthyroidism on glucose, glutamine and ketone-body metabolism in the gut of the rat. 846 60

Fibroblast cultures were used to study the effect of crude venom and six venom protein fractions (F2-F7) from Walterinnesia aegyptia on their metabolic activity. This was done by incubation of six fibroblast cultures with 10 micrograms of crude venom for 3 h at 37 degrees C. The activities of phosphofructokinase, lactate dehydrogenase, and citrate synthase were significantly lowered upon incubation with all fractions except F2. Glycogen phosphorylase activity was significantly increased, leading to a significant concurrent drop of glycogen content. This effect was only seen for fractions F3 and F5. Creatine kinase activity and cellular ATP levels rose significantly upon incubation with all venom proteins except fractions F2 and F7. Increases were seen for aspartate and alanine aminotransferases by all venom proteins except fractions F2 and F4. Incubation of cell sonicates with all the venom proteins did not significantly alter activities of any of the parameters. Thus, fibroblasts in culture under such conditions appear to mobilize glycogen, phosphocreatine, and protein for ATP production to compensate for decreased glucose.
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PMID:The effect of the desert cobra (Walterinnesia aegyptia) crude venom and its protein fractions on the metabolic activity of cultured human fibroblasts. 881 58

The rabbit kidney does not readily metabolize but synthesizes glutamine at high rates by pathways that remain poorly defined. Therefore, the metabolism of variously labeled [13C]- and [14C]glutamates has been studied in isolated rabbit kidney tubules with and without acetate. CO2, glutamine, and alanine were the main carbon and nitrogenous end products of glutamate metabolism but no ammonia accumulated. Absolute fluxes through enzymes involved in glutamate metabolism, including enzymes of four different cycles operating simultaneously, were assessed by combining mainly the 13C NMR data with a new model of glutamate metabolism. In contrast to a previous conclusion of Klahr et al. (Klahr, S., Schoolwerth, A. C., and Bourgoignie, J. J. (1972) Am. J. Physiol. 222, 813-820), glutamate metabolism was found to be initiated by glutamate dehydrogenase at high rates. Glutamate dehydrogenase also operated at high rates in the reverse direction; this, together with the operation of the glutamine synthetase reaction, masked the release of ammonia. Addition of acetate stimulated the operation of the "glutamate --> alpha-ketoglutarate --> glutamate" cycle and the accumulation of glucose but reduced both the net oxidative deamination of glutamate and glutamine synthesis. Acetate considerably increased flux through alpha-ketoglutarate dehydrogenase and citrate synthase at the expense of flux through phosphoenolpyruvate carboxykinase; acetate also caused a large decrease in flux through alanine aminotransferase, pyruvate dehydrogenase, and the "substrate cycle" involving oxaloacetate, phosphoenolpyruvate, and pyruvate.
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PMID:The rabbit kidney tubule simultaneously degrades and synthesizes glutamate. A 13C NMR study. 903 May 22

The activities of 18 enzymes involved in the intermediary and energy metabolism were measured in certain widely-spread peracarid crustaceans: 3 hypogean (Niphargus virei, Niphargus rhenorhodanensis and Stenasellus virei) and 2 epigean (Gammarus fossarum and Asellus aquaticus) ones. The activities of numerous enzymes were correlated with the known metabolic rates of the 5 species. Such rates are reduced in hypogean organisms: levels of enzymatic activity in subterranean species were 1.2 to 8.6 times lower than in epigean species for the main key regulatory enzymes involved in the Krebs cycle and glycolysis (phosphofructokinase, pyruvate kinase, hexokinase and citrate synthetase). The relative activities of phosphofructokinase, glycogen phosphorylase and hexokinase clearly indicated that glycogen was the main fuel oxidized in both epigean and hypogean organisms. A higher glycogen phosphorylase/hexokinase ratio in hypogean than in epigean crustaceans showed that subterranean species had a greater ability to function anaerobically. The presence of high activities of glutamate-pyruvate transaminase and lactate dehydrogenase in all species (and of malate dehydrogenase and fumarase in hypogean species) was indicative of a coupled fermentation of glycogen and glutamate during anaerobiosis, with lactate and alanine as end-products (as well as succinate in hypogean species). A low fructose-1,6-bisphosphatase/phosphofructokinase ratio, associated with a low level of phosphoenolpyruvate carboxykinase activity, indicated that the glycolytic pathway was active and that gluconeogenic ability was limited in epigean crustaceans. In contrast, in hypogean species, association of a higher ratio and a high level of phosphoenolpyruvate carboxykinase activity suggested a low glycolytic activity and a high gluconeogenic ability.
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PMID:The activities of enzymes associated with the intermediary and energy metabolism in hypogean and epigean crustaceans. 909 Nov 76

Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4-7 days was observed. Cells adapted to propionate still required 1-2 days before growth commenced. Incorporation of (2-13C), (3-13C) or (2H3)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The 13C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate.
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PMID:Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria. 932 32

The structure of the Escherichia coli chaperonin GroEL has been investigated by tapping-mode atomic force microscopy (AFM) under liquid. High-resolution images can be obtained, which show the up-right position of GroEL adsorbed on mica with the substrate-binding site on top. Because of this orientation, the interaction between GroEL and two substrate proteins, citrate synthase from Saccharomyces cerevisiae with a destabilizing Gly-->Ala mutation and RTEM beta-lactamase from Escherichia coli with two Cys-->Ala mutations, could be studied by force spectroscopy under different conditions. The results show that the interaction force decreases in the presence of ATP (but not of ATPgammaS) and that the force is smaller for native-like proteins than for the fully denatured ones. It also demonstrates that the interaction energy with GroEL increases with increasing molecular weight. By measuring the interaction force changes between the chaperonin and the two different substrate proteins, we could specifically detect GroEL conformational changes upon nucleotide binding.
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PMID:Atomic force microscopy detects changes in the interaction forces between GroEL and substrate proteins. 963 79

We have used citrate synthase from Thermoplasma (Tp.) acidophilum as a thermostable model system to investigate the role of hydrophobic interactions in dimer interface for maintaining high temperature stability. Three mutant enzymes were constructed by single amino acid substitutions in the interface helices: Ala97-->Ser, Ala104-->Thr, and Gly209-->Ala. All of the mutations enhanced the thermostability of Tp. citrate synthase, while improving its catalytic properties (Km, Vmax, and specific activity). The highest thermostability was achieved by the Gly209-->Ala substitution. The half-life of irreversible inactivation of the G209A mutant enzyme at 85 degreesC was about 57 min, and the midpoint of guanidinium chloride (GdmCl) induced irreversible denaturation was at 2.0 M GdmCl. Our results showed that amino acid substitutions increasing or decreasing interface hydrophobicity could further increase the thermostability of the Tp. citrate synthase. Thus, interface substitutions affecting the entropy of the unfolded state did not prove to be so critical in protein thermostabilization at higher temperatures.
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PMID:Amino acid substitutions in the subunit interface enhancing thermostability of Thermoplasma acidophilum citrate synthase. 971 38

Although glutamine synthesis has a major role in the control of acid-base balance and ammonia detoxification in the kidney of herbivorous species, very little is known about the regulation of this process. We therefore studied the influence of acetate, which is readily metabolized by the kidney and whose metabolism is accompanied by the production of bicarbonate, on glutamine synthesis from variously labelled [(13)C]alanine and [(14)C]alanine molecules in isolated rabbit renal proximal tubules. With alanine as sole exogenous substrate, glutamine and, to a smaller extent, glutamate and CO(2), were the only significant products of the metabolism of this amino acid, which was removed at high rates. Absolute fluxes through the enzymes involved in alanine conversion into glutamine were assessed by using a novel model describing the corresponding reactions in conjunction with the (13)C NMR, and to a smaller extent, the radioactive and enzymic data. The presence of acetate (5 mM) led to a large stimulation of fluxes through citrate synthase and alpha-oxoglutarate dehydrogenase. These effects were accompanied by increases in the removal of alanine, in the accumulation of glutamate and in flux through the anaplerotic enzyme pyruvate carboxylase. Acetate did not alter fluxes through glutamate dehydrogenase and glutamine synthetase; as a result, acetate did not change the accumulation of ammonia, which was negligible under both experimental conditions. We conclude that acetate, which seems to be an important energy-provider to the rabbit renal proximal tubule, simultaneously traps as glutamate the extra nitrogen removed as alanine, thus preventing the release of additional ammonia by the glutamate dehydrogenase reaction.
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PMID:Acetate stimulates flux through the tricarboxylic acid cycle in rabbit renal proximal tubules synthesizing glutamine from alanine: a 13C NMR study. 1047 67


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