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)

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.
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PMID:Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes. 24 46

Peroxisomes were isolated form derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a "Merkenschlager" cell mill (at 0 degrees C using glass beads). Catalase and urate oxidase, along with low activities of D-amino acid oxidase and L-alpha-hydroxyacid oxidase (glycollate oxidase), were associated with the peroxisomes. No catalase activity was present in glucose repressed cells. When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients. Apparently peroxisomes were destroyed as the cells became fermentative during protoplast preparation. The distribution of glyoxylate cycle enzymes was examined. Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released of present in a labile second class of peroxisomes. Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination. Citrate synthetase was not found associated with a low-density particle as had been previously reported.
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PMID:The isolation and characterization of peroxisomes (microbodies) from baker's yeast, Saccharomyces cerevisiae. 24 96

1) Albumins and globulins were prepared from dry seeds of cucumbers (Cucumis sativus) by differential extraction. The globulin fraction was analyzed by gel electrophoresis under denaturing conditions in the presence and absence of mercaptoethanol. The subunit (Mr = 54000) of the tetramer (Mr = 240000) was shown to be composed of two different peptides. Microheterogeneity rendered the exact interpretation of the analysis difficult. 2) Glyoxysomal proteins were already present in dry seeds: malate synthase, isocitrate lyase, citrate synthase, malate dehydrogenase, catalase and crotonase could be detected unequivocally. It was demonstrated that the enzymatic and immunological properties of malate synthase and isocitrate lyase were not distinguishable from that of enzymes assigned to glyoxysomes of fully developed cotyledons. 3) Homogenates prepared from seeds by cautious cell disintegration were subjected to sucrose density gradient centrifugation and yielded microbody and protein body fractions, among other things.
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PMID:Albumins, glyoxysomal enzymes and globulins in dry seeds of cucumis sativus: qualitative and quantitative analysis. 42 26

Microbodies appearing abundantly in n-alkane-grown cells of Candida tropicalis pK 233 were isolated by means of sucrose density gradient centrifugation. Electron microscopical observation showed that the microbodies isolated were intact. Localization of catalase and D-amino acid oxidase in the isolated microbodies was confirmed. Isocitrate lyase, melate synthase and NADP-linked isocitrate dehydrogenase were also located in the microbody, but malate dehydrogenase, citrate synthase, aconitase and NAD-linked isocitrate dehydrogenase were not. Neither cytochrome P-450 not NADPH-cytochrome c reductase, the components involved in the n-alkane hydroxylation system of the yeast, were detected in the microbody fraction.
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PMID:Microbody of n-alkane-grown yeast. Enzyme localization in the isolated microbody. 84 63

Treatment of rats with the vitamin B12 analogue hydroxy-cobalamin[c-lactam] (HCCL) impairs methylmalonyl-CoA mutase function and leads to methylmalonic aciduria due to intracellular accumulation of propionyl and methylmalonyl-CoA. Since accumulation of these acyl-CoAs disrupts normal cellular regulation, the present investigation characterized metabolism in hepatocytes and liver mitochondria from rats treated subcutaneously with HCCL or saline (control) by osmotic minipump. Consistent with decreased methylmalonyl-CoA mutase activity, 14CO2 production from 1-14C-propionate (1 mM) was decreased by 76% and 82% after 2-3 wk and 5-6 wk of HCCL treatment, respectively. In contrast, after 5-6 wk of HCCL treatment, 14CO2 production from 1-14C-pyruvate (10 mM) and 1-14C-palmitate (0.8 mM) were increased by 45% and 49%, respectively. In isolated liver mitochondria, state 3 oxidation rates were unchanged or decreased, and activities of the mitochondrial enzymes, citrate synthetase, succinate dehydrogenase, carnitine palmitoyltransferase, and glutamate dehydrogenase (expressed per milligram mitochondrial protein) were unaffected by HCCL treatment. In contrast, activities of the same enzymes were significantly increased in both liver homogenate (expressed per gram liver) and isolated hepatocytes (expressed per 10(6) cells) from HCCL-treated rats. The mitochondrial protein per gram liver, calculated on the basis of the recovery of the mitochondrial enzymes, increased by 39% in 5-6 wk HCCL-treated rats. Activities of lactate dehydrogenase, catalase, cyanide-insensitive palmitoyl-CoA oxidation, and arylsulfatase A in liver were not affected by HCCL treatment. Hepatic levels of mitochondrial mRNAs were elevated up to 10-fold in HCCL-treated animals as assessed by Northern blot analysis. Thus, HCCL treatment is associated with enhanced mitochondrial oxidative capacity and an increased mitochondrial protein content per gram liver. Increased mitochondrial oxidative capacity may be a compensatory mechanism in response to the metabolic insult induced by HCCL administration.
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PMID:Increased hepatic mitochondrial capacity in rats with hydroxy-cobalamin[c-lactam]-induced methylmalonic aciduria. 170 51

The effects of aging on myocardial antioxidant enzyme activity, lipid peroxidation, and other related biochemical properties were investigated in male Wistar-Furth rats at 4, 26, and 31 mo of age at rest and after an acute exercise bout. The results showed that resting heart cytosolic superoxide dismutase (CuZn SOD) activity was significantly decreased in the heart with aging (66 +/- 6.5 U/mg protein at 4 mo vs. 49 +/- 3.8 U/mg protein at 31 mo) and was elevated in all age groups after exercise. Mitochondrial Mn SOD activity was almost doubled in both 26- and 31-mo-old rats compared with that at 4 mo. Myocardial catalase and cytosolic glutathione peroxidase (GPX) activities were significantly decreased with age, whereas mitochondrial GPX was 29% higher (P less than 0.05) in 31- than 4-mo-old rats. Glutathione S-transferase activity in the heart also declined with age (P less than 0.05 at 31 mo). Malondialdehyde contents in both heart homogenate and mitochondria were significantly increased at old age. Activity of several enzymes related to myocardial energy production, e.g., citrate synthase, malate dehydrogenase, and lactate dehydrogenase, as well as myocardial protein content showed an age-related decline. These data indicate that myocardial antioxidant capacity is weakened during aging and that the compensatory increases of mitochondrial SOD and GPX may be an important mechanism in coping with free radical damage in senescent heart. Findings in the present investigation seem to support the free radical theory of aging.
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PMID:Myocardial aging: antioxidant enzyme systems and related biochemical properties. 187 97

To evaluate the peroxisomal requirement for beta-oxidation of hydroxyeicosatetraenoic acids (HETES), we tested 5-, 12- and 15-HETE oxidation in wild-type and mutant Chinese hamster ovary (CHO) cells. Mutant CHO cells contain peroxisomal ghosts, have random cytosolic localization of catalase and lack two of the enzymes necessary for peroxisomal beta-oxidation. Reverse-phase HPLC indicated that 33% of 12-HETE radioactivity was converted by wild-type CHO cells during a 2 h incubation to one major and several minor polar metabolites. Wild-type CHO cells also converted 15-HETE to one major and several minor polar metabolites. Neither 12- nor 15-HETE were converted to any metabolites by the mutant CHO cell lines, despite appreciable cellular uptake of these hydroxyeicosanoids. 5-HETE was not converted to any metabolic products by either the wild-type or the mutant CHO cells. Docosahexaenoic acid beta-oxidation was substantially reduced in the mutants as compared to the wild-type cells, palmitic acid beta-oxidation was reduced to an intermediate extent in the mutants, but octanoate beta-oxidation and citrate synthase activity were not impaired. Protein immunoblotting for mitochondrial manganese superoxide dismutase indicated a single band of identity at 20 kDa in both wild-type and mutant CHO cells. Since mutant CHO cells fail to convert 12- and 15-HETE to oxidative metabolites but contain normal mitochondrial enzymatic activities, intact peroxisomes appear to be the organelle responsible for HETE oxidation.
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PMID:Hydroxyeicosatetraenoic acid oxidation in Chinese hamster ovary cells: a peroxisomal metabolic pathway. 189 74

The present study is a follow-up to the report that dehydroepiandrosterone (DHEA) acetate treatment in rats stimulated metabolic heat production and suppressed serum triglycerides, adiposity and weight gain without affecting food intake. Activities of peroxisomal fatty acyl-coenzyme (CoA) oxidase and catalase as well as mitochondrial citrate synthase were assayed in liver tissue of 24 young adult male and female Wistar rats fed a nonpurified diet containing 0.6% DHEA (6 g/kg) for 6 wk. DHEA-treated animals gained less weight but had heavier liver weights than did the controls. Hepatic activity of fatty acyl-CoA oxidase of the experimental male and female animal was 1058 and 946% higher, respectively, than that of the controls. For catalase activity, only the female groups were different (30%). Activity of citrate synthase was not affected by DHEA. These data support the hypothesis that the inhibitory effect of DHEA on energy storage as fat is mediated, at least in part, by increased beta-oxidation of fatty acids in peroxisomes. The peroxisomal beta-oxidation pathway is uncoupled from oxidative phosphorylation; electrons are transferred directly to molecular O2 because of cycling of NAD/NADH resulting in the expenditure of chemical energy as heat.
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PMID:The effect of dehydroepiandrosterone acetate on liver peroxisomal enzyme activities of male and female rats. 295 80

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm(3) which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50-60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [(14)C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21-1.22 g/cm(3), whereas the original glyoxysomes appeared at density 1.24 g/cm(3). Electron microscopy showed that the fraction at 1.21-1.22 g/cm(3) was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.
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PMID:Localization of enzymes within microbodies. 472 5

Burton, Sheril D. (Institute of Marine Science, University of Alaska, College), Richard Y. Morita, and Wayne Miller. Utilization of acetate by Beggiatoa. J. Bacteriol. 91:1192-1200. 1966.-A proposed system which would permit acetate incorporation into four-carbon compounds without the presence of key enzymes of the citric acid cycle or glyoxylate cycle is described. In this system, acetyl-coenzyme A (CoA) is condensed with glyoxylate to form malate, which, in turn, is converted to oxaloacetate. Oxaloacetate then reacts with glutamate to produce alpha-ketoglutarate, which is subsequently converted to isocitrate. Cleavage of isocitrate produces glyoxylate and succinate. Thus, the proposed system is similar to the glyoxylate bypass in that malate is produced from glyoxylate and acetyl-CoA, but differs from both the citric acid cycle and the glyoxylate bypass, since citrate and fumarate are not involved. Fumarase, aconitase, catalase, citritase, pyruvate kinase, enolase, phosphoenolpyruvate carboxylase, lactic dehydrogenase, alpha-ketoglutarate dehydrogenase, and condensing enzyme were not detectable in crude extracts of Beggiatoa. Succinate was oxidized by a soluble enzyme not associated with an electron-transport particle. Isocitrate was identified as the sole compound labeled when C(14)O(2) was added to a reduced nicotinamide adenine dinucleotide, CO(2) generating system (crystalline glucose-6-phosphate dehydrogenase and glucose-6-phosphate) in the presence of alpha-ketoglutarate.
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PMID:Utilization of acetate by Beggiatoa. 592 51


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