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)

Citrate synthases from animal tissues were found to bind to Sepharose-"ATP." A pure preparation of citrate synthase was obtained from a crude fraction of rat heart by the specific elution of the enzyme from the Sepharose-"ATP" with the dead end complex-forming substrates, oxalacetate and CoA. The proposed mechanisms of citrate synthase, obtained from steady state kinetics, were examined in light of the elution pattern of the enzyme obtained using combinations of substrates and substrate analogs.
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PMID:Purification of and mechanism studies on citrate synthase. Use of biospecific adsorption-elution techniques. 125 79

The postnatal development of mitochondrial ATP-producing pathways and Na-K-adenosinetriphosphatase (ATPase) in the rat medullary thick ascending limb of Henle (MTAL) was studied by measuring the activities of 3-ketoacid-CoA transferase, fumarase, citrate synthase, and Na-K-ATPase in microdissected MTAL of 16, 21, and 30-day-old pups and in adults. The role of adrenal steroids in the development of these four markers was also investigated by studying 21-day-old rats adrenalectomized on day 16 and given dexamethasone or aldosterone or NaCl injections from day 16 to day 21. There were large and correlated increases in the activities of the oxidative enzymes in the MTAL of control rat kidneys between 16 and 30 days after birth; Na-K-ATPase activity in the MTAL also greatly increased during the same period. Adrenalectomy completely prevented the developmental increases in MTAL oxidative enzymes and Na-K-ATPase; dexamethasone restored the development of all four enzymes, whereas aldosterone had no effect. We conclude that the postnatal maturation of Na+ reabsorption functions in MTAL cells involves coordinated increases in the capacity to produce ATP by oxidative metabolism and in Na-K-ATPase activity. This maturation process is probably triggered by the rise in circulating glucocorticoids that occurs during the weaning period.
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PMID:Coordinate development of oxidative enzymes and Na-K-ATPase in thick ascending limb: role of corticosteroids. 132 5

Citrate synthase complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CM-CoA), are believed to mimic those with the activated form of acetyl-CoA. The X-ray structure [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213] of the ternary complex of the enzyme, oxaloacetate, and CMCoA has been used as the basis for a proposal that a neutral enol of acetyl-CoA is that activated form. Since the inhibitor carboxyl has a pKa of 3.90, analogy with an enolic acetyl-CoA intermediate leads to the prediction that a proton should be taken up from solution upon formation of the analog complex so that the transition-state analog carboxyl is protonated when bound. We have obtained evidence in solution for this proposal by comparing the isoelectric points and the pH dependence of the dissociation constants of the ternary complexes of the pig heart enzyme with the neutral ground-state analog inhibitor, acetonyl-CoA (KCoA), and the anionic transition-state analog inhibitor (CMCoA) and by studying the NMR spectra of the transition-state analog complexes of allosteric (Escherichia coli) and nonallosteric (pig heart) enzymes. The pH dependence of the dissociation constant of the ground-state analog indicates no proton uptake, while that for the transition-state analog indicates that 0.55 +/- 0.04 proton is taken up when the analog binds to the citrate synthase-oxaloacetate binary complex. The overall charges of ternary complexes of the pig heart enzyme with the transition-state and ground-state analog inhibitors are the same, as monitored by their isoelectric points.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proton uptake accompanies formation of the ternary complex of citrate synthase, oxaloacetate, and the transition-state analog inhibitor, carboxymethyl-CoA. Evidence that a neutral enol is the activated form of acetyl-CoA in the citrate synthase reaction. 132 22

Acetyl-CoA enol has been proposed as an intermediate in the citrate synthase (CS) reaction with Asp375 acting as a base, removing a proton from the methyl carbon of acetyl-CoA, and His274 acting as an acid, donating a proton to the carbonyl [Karpusas, M., Branchaud, B., & Remington, S.J. (1990) Biochemistry 29, 2213]. CS-oxaloacetate (OAA) complexes with the transition-state analog inhibitor, carboxymethyl-CoA (CMCoA), mimic those with acetyl-CoA enol. Asp375 and His274 interact intimately with the carboxyl of the bound inhibitor. While enzymes in which these residues have been changed to other amino acids have very low catalytic activity, we find that they retain their ability to form complexes with substrates and the transition-state analog inhibitor. In comparison with the value of the chemical shift of the protonated CMCoA carboxyl in acidic aqueous solutions or its value in the wild-type ternary complex, the values in the Asp375 mutants are unusually low. Model studies suggest that these low values result from complete absence of one hydrogen bond partner for the Gly mutant and distortions in the active site hydrogen bond systems for the Glu mutant. The high affinity of Asp375Gly-OAA for CMCoA suggests that the unfavorable proton uptake required to stabilize the CMCoA-OAA ternary complex of the wild-type enzyme [Kurz, L.C., Shah, S., Crane, B.R., Donald, L.J., Duckworth, H.W., & Drysdale, G.R. (1992) Biochemistry (preceding paper in this issue)] is not required by this mutant; the needed proton is most likely provided by His274. This supports the proposed role of His274 as a general acid.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catalytic strategy of citrate synthase: effects of amino acid changes in the acetyl-CoA binding site on transition-state analog inhibitor complexes. 132 23

The purpose of the present study was to determine the effects of low-frequency electrical stimulation (LFES) on the skeletal muscle metabolic profile of men and women. The knee extensor muscles of sedentary men (N = 16) and women (N = 10) were submitted to 3 h.d-1 of 8-Hz neuromuscular electrical stimulation with the use of a portable stimulator (Respond II, Medtronic), 6 d.wk-1 for 6 wk. Enzyme activity levels of creatine kinase (CK), hexokinase (HK), glyceraldehydephosphate dehydrogenase (GAPDH), 3-hydroxyacyl CoA dehydrogenase (HADH), citrate synthase (CS), phosphofructokinase (PFK), and cytochrome c oxidase (COX) were determined in vastus lateralis muscle samples taken before and after the LFES protocol. The analyses of variance revealed no change in CK and in GAPDH. However, a small decrease in PFK activity, the rate-limiting enzyme of glycolysis, was observed in female (8%) and in male subjects (10%), but it reached significance in males only (P < 0.05). The activity level of HK, a regulatory enzyme of the skeletal muscle glucose phosphorylation (HK), increased significantly in female subjects only (36%; P < 0.01) in response to the stimulation protocol. Activity level of marker enzymes of the Krebs cycle (CS) and of the electron-transfert chain (COX) significantly increased in males (18% and 16%; P < 0.05) as well as in females (31% and 19%; P < 0.05). Increment in the marker enzyme activity of the fatty acid oxidation (HADH) was significant in female subjects (30%; P < 0.01) and, although significant, rather modest in male subjects (12%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Electrical stimulation-induced changes in skeletal muscle enzymes of men and women. 133 93

Muscle strength and muscle morphology have been studied three times during a period of 11 yr in nine elderly men. On the last occasion the average age was 80.4 (range 79-82) yr. Body cell mass decreased by 6% and muscle strength for knee extension, measured by means of isometric and concentric isokinetic (30-60 degrees/s) recordings, declined by 25-35% over the 11-yr period. Between 76 and 80 yr of age only the isokinetic strength for 30 degrees/s decreased significantly. Muscle fiber composition in the vastus lateralis did not change between 69 and 76 yr of age, but there was a significant reduction in the proportion of type IIb fibers from 76 to 80 yr. The decrease in type II fiber areas was not significant between 69 and 76 yr of age (as in a larger sample from the same population), but a significant increase in both type I and type II fiber areas was recorded from 76 to 80 yr of age and biceps brachii showed similar tendencies. In the same period, the enzymatic activities of myokinase and lactate dehydrogenase subsided in the vastus lateralis, but there was no change for triose phosphate dehydrogenase, 3-hydroxy-CoA-dehydrogenase, and citrate synthase. The muscle fiber hypertrophy in this group of elderly men with maintained physical activity between 76 and 80 yr of age is interpreted as a compensatory adaptation for the loss of motor units. In addition, the adaptation with respect to oxidative capacities seems to be maintained at this age.
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PMID:Compensatory muscle fiber hypertrophy in elderly men. 140 42

The individual anaerobic threshold (Th(an)) is the highest metabolic rate at which blood lactate concentrations can be maintained at a steady-state during prolonged exercise. The purpose of this study was to test the hypothesis that training at the Th(an) would cause a greater change in indicators of training adaptation than would training "around" the Th(an). Three groups of subjects were evaluated before, and again after 4 and 8 weeks of training: a control group, a group which trained continuously for 30 min at the Th(an) intensity (SS), and a group (NSS) which divided the 30 min of training into 7.5-min blocks at intensities which alternated between being below the Th(an) [Th(an) -30% of the difference between Th(an) and maximal oxygen consumption (VO2max)] and above the Th(an) (Th(an) +30% of the difference between Th(an) and VO2max). The VO2max increased significantly from 4.06 to 4.27 l.min-1 in SS and from 3.89 to 4.06 l.min-1 in NSS. The power output (W) at Th(an) increased from 70.5 to 79.8% VO2max in SS and from 71.1 to 80.7% VO2max in NSS. The magnitude of change in VO2max, W at Th(an), % VO2max at Th(an) and in exercise time to exhaustion at the pretraining Th(an) was similar in both trained groups. Vastus lateralis citrate synthase and 3-hydroxyacyl-CoA-dehydrogenase activities increased to the same extent in both trained groups. While all of these training-induced adaptations were statistically significant (P < 0.05), there were no significant changes in any of these variables for the control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Adaptations to training at the individual anaerobic threshold. 142 31

Coenzyme A without an acyl-thioester (CoASH) is required for numerous cellular reactions, and sequestration of CoASH as acyl-CoAs may impair metabolic function. Increased total CoA protects the cell from acyl-CoA accumulation, and enhanced CoA biosynthesis may represent a compensatory response in metabolic disease. To test the hypothesis that cellular CoA is redistributed from the cytosol to the mitochondria in response to mitochondrial acyl-CoA accretion, the subcellular distribution of hepatic CoA was determined by differential centrifugation and measurement of the mitochondrial marker enzyme citrate synthase. Liver from control, clofibrate-treated and hydroxycobalamin[c-lactam] (HCCL)-treated rats were used. Clofibrate increased total hepatic CoA concentration 2.2-fold, whereas HCCL (which causes inhibition of L-methylmalonyl-CoA mutase and consequent propionyl- and methylmalonyl-CoA accumulation) increased it threefold. However, clofibrate did not affect the percentage of total CoA in the mitochondria (control: 44 +/- 3%, clofibrate: 49 +/- 5%), and HCCL-treatment induced a marked redistribution of CoA into the mitochondria (HCCL: 78 +/- 8%). Redistribution of total CoA was also induced acutely by incubation of hepatocytes from control rats with 10 mmol/L propionate. Thus, redistribution of the cellular CoA pool can help maintain CoASH availability as mitochondrial acyl-CoA accumulation occurs and may be an important compensatory response to metabolic injury.
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PMID:Rat hepatic coenzyme A is redistributed in response to mitochondrial acyl-coenzyme A accumulation. 143 50

1. Limited proteolysis of citrate synthase from Sulfolobus solfataricus by trypsin reduced the rate of the overall reaction (acetyl-CoA + oxaloacetate + H2O----citrate + CoASH) to 4% but did not affect the hydrolysis of citryl-CoA. Experimental results indicate that a connecting link between the enzyme's ligase and hydrolase activity becomes impaired specifically on treatment with trypsin. Other proteolytic enzymes like chymotrypsin and subtilisin inactivated catalytic functions of citrate synthase, ligase and hydrolase, equally well. 2. Tryptic hydrolysis occurs at the N-terminal region of citrate synthase, but a study by SDS/PAGE revealed no difference in molecular mass between native and proteolytically nicked citrate synthase. The peptide removed from the enzyme by trypsin, therefore, contains less than about 15 amino acid residues. 3. The Km values of the substrates for both native and nicked enzyme were identical, as was the state of aggregation (dimeric) of the two enzyme species. These could be separated by affinity chromatography on Blue-Sepharose and differentiated by their isoelectric points (pI = 6.68 +/- 0.08 and pI = 6.37 +/- 0.03 for native citrate synthase and the large tryptic peptide, respectively) as well as by the N-terminus which is blocked in the native enzyme only. 4. Edman degradation of the large tryptic fragment yielded the N-terminal sequence GLEDVYIKSTSLTYIDGVNGVLRY, which is 71% identical to the N-terminal region (positions 9-32) of citrate synthase from Thermoplasma acidophilum. 5. The conversion of citrate synthase into essentially a citryl-CoA hydrolase is considered the consequence of a conformational change thought to occur on tryptic removal of the N-terminal small peptide.
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PMID:Conversion, by limited proteolysis, of an archaebacterial citrate synthase into essentially a citryl-CoA hydrolase. 152 37

Much has been learned about FACES of the endoplasmic reticulum since its discovery in the early 1960s. FACES consists of four component reactions, requires the fatty acid to be activated in the form of a CoA derivative, utilizes reducing equivalents in the form of NADH or NADPH, is induced by a fat-free diet, resides on the cytoplasmic surface of the endoplasmic reticulum, appears to function in concert with the desaturase system and appears to exist in multiple forms (either multiple condensing enzymes connected to a single pathway or multiple pathways). FACES has been found in all tissues investigated, namely, liver, brain, kidney, lung, adrenals, retina, testis, small intestine, blood cells (lymphocytes and neutrophils) and fibroblasts, with one exception--the heart has no measurable activity. Yet, much more needs to be learned. The critical, inducible and rate-limiting condensing enzyme has resisted solubilization and purification; the purification of the other components has met with limited success. We know nothing about the site of synthesis of each component of FACES. How is each component enzyme integrated into the endoplasmic reticulum membrane? Is there a single mRNA directing synthesis of all four components or are there four separate mRNAs? How are elongation and desaturation coordinated? What is (are) the physiological regulator(s) of FACES--ADP, AMP, IP3, G-proteins, phosphorylation, CoA, Ca2+, cAMP, none of these? The molecular biology of FACES is only in the fetal stage of development. We are only scratching the surface--it is an undiscovered country.
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PMID:The fatty acid chain elongation system of mammalian endoplasmic reticulum. 164 95


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