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)

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

Genomic libraries of Mycobacterium leprae DNA partially digested with Pst I were constructed in the expression vector pYA626, which contains the promoter region from the Streptococcus mutans gene encoding aspartate beta-semialdehyde dehydrogenase, which is very efficiently expressed in Escherichia coli. We have detected several clones that complement a mutation in the citrate synthase gene of E. coli. Southern blot analysis demonstrated that the complementing DNA was M. leprae DNA. Sodium dodecyl sulfate/polyacrylamide gel analysis of polypeptides produced by minicells containing the citrate synthase-complementing recombinant molecules demonstrated the production of a 46-kDa polypeptide. When the citrate synthase-complementing fragment was cloned in pYA626 in the reverse orientation, the recombinant molecule was no longer able to complement the mutation in the citrate synthase gene and no longer produced the 46-kDa polypeptide. When the DNA fragment was cloned in the Pst I site of pHC79, so as to allow expression from the beta-lactamase promoter, the resulting recombinant failed to complement the mutation in the E. coli citrate synthase gene yet still produced the 46-kDa polypeptide, but in one-fourth the amount than when expressed from the S. mutans asd promoters. This demonstrates that M. leprae translational sequences can be recognized by E. coli translational machinery. Promoter expression vectors can be used to obtain expression of protein antigens to be used for early diagnosis of leprosy or components of a vaccine and proteins that are targets of potential antileprosy drugs.
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PMID:Expression of Mycobacterium leprae genes from a Streptococcus mutans promoter in Escherichia coli K-12. 286 92

A cDNA that encodes pig citrate synthase (PCS) was inserted into a plasmid T7 vector and was expressed in an E. coli gltA mutant. Up to 10 mg of purified PCS was obtained from 2 liters of E. coli. The mammalian protein produced in E. coli comigrated with the enzyme purified from pig heart on a SDS-polyacrylamide gel (SDS-PAGE) with an Mr of 50,000, and reacted with a polyclonal antibody directed against pig heart citrate synthase. The Vmax and Km of the expressed PCS were indistinguishable from those of the pig heart enzyme. The PCS produced in E. coli did not contain the trimethylation modification of Lys 368, characteristic of the pig heart enzyme. These data suggest that the PCS protein produced in E. coli is catalytically similar to the enzyme purified from pig heart and methylation of Lys 368 is not essential for catalysis.
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PMID:Characterization of mutant TMK368K pig citrate synthase expressed in and isolated from Escherichia coli. 314 69

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.
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PMID:Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate. 328 13

3-Hydroxyacyl coenzyme A (CoA) dehydrogenase-binding protein was solubilized from inner mitochondrial membrane by using taurodeoxycholate at high ionic strength. The binding protein was isolated from the suspension using 3-hydroxyacyl-CoA dehydrogenase affinity chromatography. The protein eluted from the affinity column had a molecular weight of approximately 150,000, as determined by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protein is a dimer consisting of 69,000 and 71,000 molecular weight subunits. The enzyme binding capacity of this protein was tested with a polyethylene glycol precipitation method: 0.5 mg of enzyme could be precipitated together with 1 mg of binding protein, showing that 1 mol of binding protein binds 1 mol of enzyme. This protein had no affinity toward malic dehydrogenase, citrate synthase, and fumarase. The approximately 2-fold increase in the 3-hydroxyacyl-CoA dehydrogenase activity when it was measured in the presence of the binding protein is additional evidence of enzyme-binding protein interaction. When incorporated into liposomes, the binding protein retained its ability to bind 3-hydroxyacyl-CoA dehydrogenase, but did not bind malic dehydrogenase, citrate synthase, and fumarase. These results suggest that the protein isolated by us has a specific function in anchoring a beta-oxidation enzyme to the matrix surface of the mitochondrial membrane.
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PMID:Isolation and characterization of 3-hydroxyacyl coenzyme A dehydrogenase-binding protein from pig heart inner mitochondrial membrane. 377 31

Citrate synthase from Acinetobacter calcoaceticus was subjected to proteolysis with subtilisin. Although the enzyme proved relatively resistant to inactivation by this treatment, SDS-polyacrylamide gel electrophoresis clearly revealed breakdown of the citrate synthase to smaller fragments. The regulatory responses of the native enzyme to inhibition by NADH and re-activation by AMP were retained on proteolysis, indicating that the fragments bind tightly to each other and preserve the overall cooperative molecular interactions.
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PMID:Proteolysis of acinetobacter citrate synthase by subtilisin. 633 74

A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit Mr was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.
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PMID:Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene. 635 85

Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondria matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the percipitates with concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linkers enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzyme are preferentially located near the membrane.
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PMID:Cross-linking of mitochondrial matrix proteins in situ. 640 45

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.
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PMID:Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme. 641 81

Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.
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PMID:Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis. 751 28


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