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 possible induction of renal citrate synthase (E.C. 4.1.3.7) by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 microgram/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968. Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on the KmS for acetyl-CoA and oxalacetate but augmented Vmax of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation. The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 microgram/100 g body wt) or the diluent, and simultaneously with 3H or 35S methionine (500 muCi/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 microcram/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either 3H- or 35S-methionine at 20 degrees for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70--80 microgram/100 g body wt) or spirolactone (SC-26304) (80 microgram/100 g body wt). An equimolar dose of dexamethasone (0.8 microgram/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.
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PMID:Induction of citrate synthase by aldosterone in the rat kidney. 35 85

A combination of equilibrium ultracentrifugation and polyacrylamide gel electrophoresis techniques has been used to establish the quaternary structure of citrate synthase from acetate-grown Escherichia coli K12 3000. In polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS), the pure enzyme showed one major band whose mobility was consistent with a molecular weight of 46,000 plus or minus 2000 g/mol, and a little material of 87,000 plus or minus 5000 g/mol. When first cross-linked with dimethyl suberimidate and then submitted to electrophoresis in SDS, citrate synthase showed six bands, in widely different amounts, whose apparent molecular weights were almost integral multiples of 47,000 g/mol. The dimer was the major product of the cross-linking procedure. In 6 M guanidine HCl at pH 7.0, citrate synthase behaved as a single component in high-speed sedimentation equilibrium experiments, with a weight average molecular weight of 43,400 plus or minus 300 g/mol. The molecular weight of native citrate synthase was investigated by high-speed sedimentation equilibrium ultracentrifugation under different conditions of pH and KCl concentration. In 0.02 M Tris-Cl at pH 7.0 and 7.8, the enzyme was a mixture of oligomers, with species ranging from monomer (47,000 g/mol) to greater than decamer being present. At pH 9.0, only dimer was seen (94,000 g/mol). Large aggregates were present at pH 10.0. The addition of small amounts of KCl, a potent activator of the enzyme, simplified the mixture of oligomers considerably at pH 7.8. A detailed analysis of the data with 0.05 M KCl indicated that dimer and hexamer were the only species present, with marked nonideality. Increasing the KCl concentration to 0.10 M converted all the enzyme to hexamer. The amino acid composition of E. coli citrate synthase was presented. Taken together with peptide mapping experiments of others (J. A. Wright and B. D. Sanwal (1971), J. Biol. Chem. 246 1689), it indicates that the subunits have all the same or very similar amino acid sequences. The dansylation method revealed only methionine at the N-termini of the citrate synthase polypeptide chains. Citrate synthase from E. coli thus resembles the enzyme from eukaryotes in that it consists of subunits weighing just under 50,000 g/mol, although these subunits are more highly aggregated in the bacterial enzyme under most conditions. This conclusion is in disagreement with that of Wright and Sanwal (1971, see above), who reported a subunit size of 62,000 g/mol.
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PMID:The quaternary structure of citrate synthase from Escherichia coli K12. 109 Dec 85

[35S]Methionine-labeled porcine heart citrate synthase (used here as a positive control) and rat liver carnitine palmitoyltransferase II (CPT II) were generated by in vitro transcription and translation of their cDNA constructs in appropriate Bluescript plasmids. Each product was imported into rat liver mitochondria in an energy-dependent manner to yield an immunoprecipitable protein of smaller size that comigrated with the corresponding purified enzyme. The size shift occurring with citrate synthase was consistent with the removal of the postulated 27-amino acid leader peptide. To determine the amino terminus of mature CPT II, [35S]methionine- or [3H]leucine-labeled material (after import and processing) was subjected to Edman degradation, followed by counting of the radioactivity released on each cycle. The results established that the precursor targeting peptide was cleaved between leucine 25 and serine 26 in the previously deduced amino acid sequence. Taken in conjunction with the recent report of Finocchiaro et al. (Finocchiaro, G., Taroni, F., Rocchi, M., Martin, A. L., Colombo, I., Tarelli, G. T., and DiDonato, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 661-665), the present results establish three key points concerning the rat and human forms of CPT II. First, it appears that in both species the initial translation product contains 658 amino acids and, upon mitochondrial import, is reduced in length by 25 residues through cleavage at an identical site. Second, the difference in electrophoretic mobility between the two mature proteins (documented earlier) presumably reflects either anomalous behavior of one of them on polyacrylamide gels or differential covalent modification. Finally, the recent suggestion by Brady et al. (Brady, P. S., Liu, J. S., Park, E. A., Hanson, R. W., and Brady, L. J. (1991) FASEB J. 5, A817) that our CPT II cDNA construct is incomplete in the 5'-coding region is refuted.
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PMID:Mitochondrial import and processing of rat liver carnitine palmitoyltransferase II defines the amino terminus of the mature protein. Possibility of differential modification of the rat and human isoforms. 186 64

A Mycobacterium smegmatis PstI library was constructed by cloning these fragments downstream from the lac promoter of the expression vector pHG171. Three identically sized clones were isolated by complementation of an Escherichia coli strain (chi 2338) deficient in citrate synthase. One insert (pBL265) was used in hybridization experiments with DNA from E. coli and M. smegmatis and it was demonstrated that the clones were indeed from M. smegmatis. The transcription of the M. smegmatis citrate synthase gene in E. coli relied upon the lac promoter. In translation experiments performed in vitro pBL265 gave rise to a novel protein of about 42 kDa. This band was not seen in 'opposite-orientation' subclones. Various subclones in which the 5'-end was shortened nevertheless complement E. coli chi 2338 and produce the 42 kDa protein. This demonstrates that the M. smegmatis citrate synthase gene uses its own ribosome-binding site in E. coli. The relevant 1.8 kb of the 2.8 kb insert was sequenced. A consensus E. coli ribosome-binding site was found centred precisely 10 bp upstream of the methionine codon. Other interesting features revealed by the sequence are discussed. Citrate synthase activity was assayed in vitro and the mycobacterial enzyme was found to be similar to those of the Gram-positive bacteria.
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PMID:Citrate synthase from Mycobacterium smegmatis. Cloning, sequence determination and expression in Escherichia coli. 188 31

Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase. 304 87

The sequence of 1895 base pairs of Acinetobacter anitratum genomic DNA, containing the structural gene for the allosteric citrate synthase of that Gram-negative bacterium, is presented. The sequence contains an open reading frame of 424 codons, the 5' end of which is the same as the N-terminal sequence of A. anitratum citrate synthase, less the initiator methionine. The inferred amino acid sequence of the enzyme is about 70% identical with that of citrate synthase from Escherichia coli, which like the A. anitratum enzyme is sensitive to allosteric inhibition by NADH. There is also a more distant homology with the nonallosteric citrate synthases of pig heart and yeast. The gene contains sequences that strongly resemble those found in E. coli promoters, an E. coli type of ribosomal binding site, and a hyphenated dyad sequence at the 3' end of the gene which resembles the rho-independent terminators found in some E. coli genes. The plasmid clone containing the A. anitratum citrate synthase gene pLJD1, originally isolated because it hybridized with the cloned E. coli citrate synthase gene under conditions of reduced stringency, produces large amounts of A. anitratum citrate synthase in an E. coli host which lacks citrate synthase. This work completes proof of the hypothesis that the three major kinds of citrate synthases are formed of similar subunits, although their functional properties are different.
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PMID:Expression and base sequence of the citrate synthase gene of Acinetobacter anitratum. 344 26

The citrate synthase of yeast was purified to homogeneity and shown to have a subunit molecular weight of 52,000. Antibodies were prepared to it in rabbits. Translation of total yeast mRNA using a reticulocyte lysate showed that [3H]leucine was incorporated into a protein precipitable by rabbit anti-yeast citrate synthase with a molecular weight of about 54,000. No incorporation of [35S]methionine into an anti-citrate synthase precipitable protein could be detected in a similar experiment. In vivo labeling of cells with 35SO4 did not result in the labeling of citrate synthase.
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PMID:In vitro translation of mRNA for yeast citrate synthase. 680 68

Using the selective membrane-solubilizing properties of digitonin and a rapid centrifugation method to separate cytoplasmic and mitochondrial components, the metabolic state of mitochondrial glutathione was investigated in isolated rat hepatocytes. Two pools of GSH were released from hepatocytes incubated with increasing concentrations of digitonin. The largest pool (about 85% of cellular total) was released simultaneously with lactate dehydrogenase, the other pool with citrate synthase, indicating cytoplasmic and mitochondrial locations, respectively. The t1/2 of the mitochondrial pool was estimated by linear regression analysis to be 30 +/- 3 h, while the cytoplasmic pool turned over with a t1/2 of about 2 +/- 0.1 h. The rate of incorporation of [35S]methionine or cysteine into the cytoplasmic pool of GSH, when corrected for turnover, was 15 times greater than into the mitochondrial pool. Mitochondrial GSH was not depleted after 60 min with 185 microM diethyl maleate with or without 75 microM bis-1,3-(2-chloroethyl)-1-nitrosourea, a specific inhibitor of glutathione reductase, whereas cytoplasmic levels were reduced to 40% and 10% of control values, respectively. In vivo experiments, using L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid to inactive gamma-glutamyl transpeptidase to limit cysteine formation from plasma GSH, demonstrated that in the absence of label reincorporation, liver glutathione exhibits a biphasic turnover. The rates of decay (half-lives) and percentages of total GSH under these conditions correlate well with the half-lives and pool distribution seen in the mitochondrial and cytoplasmic populations of GSH found in the isolated hepatocytes.
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PMID:Status of the mitochondrial pool of glutathione in the isolated hepatocyte. 706 8

The detailed proof of the 437-residue amino acid sequence (Mr 48,969) of porcine heart citrate synthase (EC 4.13.7) is described. The S-carboxymethylated protein has been cleaved at methionine (cyanogen bromide) and arginine (trypsin digest of citraconylated enzyme) residues to yield 14 and 17 major peptides, respectively. Peptides were initially fractionated by gel filtration, and those useful for sequence analysis were purified by high-performance liquid chromatography. Sequence analyses were performed on these primary peptides and on subpeptides generated by cleavage with the bromine adduct of 2-[(2-nitrophenyl)sulfenyl]-3-methylindole, Staphylococcus aureus V8 protease, trypsin, chymotrypsin, or acid. The overall sequence was confirmed by analyzing products of cleavage by hydroxylamine, acid, and subtilisin. A novel feature of the sequence is the identification of trimethyllysine at residue 368.
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PMID:Complete amino acid sequence of porcine heart citrate synthase. 709 27

The effect of the chronic intramuscular administration of some agents related to the S-adenosyl-L-methionine system on the hyperammonemia syndrome was evaluated. This experimental syndrome was induced in the rat by intraperitoneal administration of high doses of ammonium acetate (33, 100 and 300 mg/kg/day, 6 days a week for 80 days) followed by the assay of the activities of some cerebral enzymes involved in energy transduction. The enzymatic activities studied in the homogenate and in the mitochondrial fractions of brain tissue were: lactate dehydrogenase, citrate synthase, malate dehydrogenase, total NADH-cytochrome c reductase and cytochrome oxidase. All three doses of ammonium acetate induced significant modifications in the cerebral enzymatic activities. These doses reduced the activity of the total NADH-cytochrome c reductase both in the homogenate and in the mitochondrial fraction. On the other hand the activity of malate dehydrogenase was reduced limited to the two lower doses in the homogenate only. The simultaneous daily treatment (i.m.) with equimolar doses of substances involved in the S-adenosyl-L-methionine system (adenosine, methionine and S-adenosyl-L-methionine) did not cause any significant modification of the cerebral enzymatic activities associated with the administration of ammonium acetate at the three dose levels, thus confirming our previous results.
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PMID:Cerebral enzymatic activities during chronic hyperammonemia and treatment with S-adenosyl-L-methionine, adenosine and methionine in the rat. 725 Mar 59


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