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)

Two types of citrate synthase (CS) have been purified from Pseudomonas aeruginosa, a 'large' form (CSI) and a 'small' form (CSII). The M(r)s of the CSI and CSII isoenzymes were determined to be 240,000 +/- 16,000 (mean +/- S.E.M.) and 80,300 +/- 3800 respectively. Chemical cross-linking of the native enzymes with either dimethyl suberimidate or glutaraldehyde followed by electrophoretic analysis by SDS/PAGE showed that CSI is a hexamer and CSII is a dimer. SDS/PAGE showed that CSI and CSII each consist of a single subunit type, of M(r) 42,000 +/- 2000 and M(r) 36,500 +/- 2000 respectively. CSI and CSII were also shown to be distinct kinetically, immunologically and in terms of their regulatory properties. It is suggested that the CS isoenzymes are products of different structural genes.
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PMID:Purification and characterization of citrate synthase isoenzymes from Pseudomonas aeruginosa. 762 13

A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.
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PMID:Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas. 890 35

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5'-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast lambdaEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae.
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PMID:Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae. 921 8

Citrate synthase which condenses acetyl-CoA and oxaloacetate to citrate was purified from Drosophila melanogaster. Some physicochemical as well as enzymatical properties were investigated. The optimum pH and temperature were pH 8.0-9.0 and 45 degrees C, respectively. The molecular weight of the enzyme was determined as 81,000 Da by gel filtration and the purified active enzyme consisted of two identical subunits which had a molecular mass of 48,700 on SDS-PAGE. Homogeneity of the purified enzyme was confirmed by SDS-PAGE and also by N-terminal amino acid sequence analysis. The Michaelis constants (K(m)) of the enzyme for acetyl-CoA and oxaloacetate were 6.7 microM and 3.1 microM, respectively. Kinetic studies showed that citrate synthase follows the concerted mechanism which forms a ternary complex. Propionyl-CoA, ATP, and intermediates of the TCA cycle, succinyl-CoA and alpha-ketoglutarate, behaved as inhibitors in vitro. Using pig and chicken heart enzymes for comparison, we found similarities at the N-terminal region. However, in the Ouchterlony immunodiffusion test, the polyclonal antibody raised against Drosophila citrate synthase did not show any crossreaction with pig, chicken or pigeon enzymes.
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PMID:Characterization of citrate synthase purified from Drosophila melanogaster. 938 45

We report direct experimental evidence that human alphaB-crystallin, a member of the small heat shock protein family, actively participates in the refolding of citrate synthase (CS) in vitro. In the presence of 3.5 mM ATP, CS reactivation by alphaB-crystallin was enhanced approximately twofold. Similarly, 3.5 mM ATP enhanced the chaperone activity of alphaB-crystallin on the unfolding and aggregation of CS at 45 degrees C. Consistent with these findings, cell viability at 50 degrees C was improved nearly five orders of magnitude in Escherichia coli expressing alphaB-crystallin. SDS/PAGE analysis of cell lysates suggested that alphaB-crystallin protects cells against physiological stress in vivo by maintaining cytosolic proteins in their native and functional conformations. This report confirms the action of alphaB-crystallin as a molecular chaperone both in vitro and in vivo and describes the enhancement of alphaB-crystallin chaperone functions by ATP.
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PMID:ATP-enhanced molecular chaperone functions of the small heat shock protein human alphaB crystallin. 944 75

To investigate effects of sustained activity on major phenotypic properties, the left extensor digitorum longus muscle of young (15 wk) and aging (101 wk) male Brown Norway rats was subjected to 50 days of chronic low-frequency stimulation (CLFS; 10 Hz, 10 h/day). The contralateral muscle served as control. Changes in metabolic enzymes were analyzed by using glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase as reference enzymes of glycolysis and by using citrate synthase and 3-hydroxyacyl-CoA dehydrogenase as mitochondrial enzymes representative of aerobic-oxidative metabolism. Myosin heavy chain (MHC) isoforms were analyzed by SDS-PAGE. No differences existed between the enzyme activity profiles of control muscles from young and aging rats. CLFS induced similar increases in mitochondrial enzymes, as well as similar decreases in glycolytic enzymes. Although the MHC composition of the control muscles in the aging rats displayed a shift toward slower isoforms, the ultimate changes induced by CLFS led to nearly identical MHC phenotypes in both young and aging rats. These results demonstrate an unaltered adaptability of skeletal muscle to increased neuromuscular activity in the aging rat.
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PMID:Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats. 968 17

Different severe disorders of cytochrome c oxidase (COX) have been described in children, but only the defects with autosomal inheritance are suitable for prenatal diagnosis. To perform prenatal diagnosis of fatal infantile COX deficiency a complex approach has been used which combined determination of the genetic origin of the defect, and detailed analysis of the function, content and subunit composition of the enzyme in cultured fetal cells. The tissues and cultured fibroblasts of the patient with Leigh's syndrome showed a COX deficiency of systemic character. The decrease of COX activity to 5-11 per cent was accompanied by proportionally decreased content of the assembled COX enzyme. With the help of transmitochondrial cybrids derived from patient fibroblasts it was proven that the COX defect was of nuclear origin. In a successive pregnancy, the function of oxidative phosphorylation (OXPHOS) was analysed in cultured amniocytes by substrate-stimulated ATP production and COX activity was compared with the activity of citrate synthase. The amount and composition of OXPHOS complexes was estimated by two-dimensional (Blue Native/SDS) polyacrylamide gel electrophoresis and was verified immunochemically with specific antibodies. Three independent lines of evidence provided us with reliable data on the function of COX and OXPHOS in fetal cells which were sufficient to rule out the expected enzymatic defect within three weeks after amniocentesis.
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PMID:Complex approach to prenatal diagnosis of cytochrome c oxidase deficiencies. 1041 72

The 90-kDa heat shock protein (HSP90) acts as a specific molecular chaperone in the folding and regulates a wide range of associated proteins such as steroid hormone receptors. It is known that HSP90 possesses two different chaperone sites, both in the N- and C-domains, and that the chaperone activity of HSP90 is blocked by binding of geldanamycin (GA) to the N-domain, the same as the ATP-binding site. Here we show that Cisplatin [cis-diamminedichloroplatinum (II), CDDP], an antineoplastic agent, associates with HSP90 and reduces its chaperone activity. In order to analyse the binding proteins, bovine brain cytosols were applied to a CDDP-affinity column and binding proteins were eluted by CDDP. In the elutants, only 90-kDa protein bands were detected on SDS/PAGE, and the protein was cross-reacted with the anti-HSP90 antibody on immunoblotting. No protein bands were detected in the elutants from the control column on SDS/PAGE. These results indicated that CDDP has a high affinity for HSP90. On CD spectrum analysis, the binding of CDDP to HSP90 resulted in a conformational change in the protein. Although HSP90 inhibited the aggregation of citrate synthase as a molecular chaperone in vitro, the activity was suppressed almost completely in the presence of CDDP. Mg/ATP has an influence on the chaperone activity to some extent. The CDDP binding region of HSP90 is near the C-terminal which is quite different from the GA-binding site. Our results suggest that the chaperone activity of HSP90 may be inhibited by the binding of CDDP or GA by different mechanisms.
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PMID:A novel chaperone-activity-reducing mechanism of the 90-kDa molecular chaperone HSP90. 1052 51

It has been reported that immunosuppressant cyclosporin A or FK506 binds to immunophilins in the cell and that these immunophilins make a complex with molecular chaperones HSP70 or HSP90. Although mizoribine has been used clinically as an immunosuppressant, immunophilins of the agent have not yet been fully understood. We investigated their specific binding proteins using mizoribine affinity column chromatography and porcine kidney cytosols. By increasing mizoribine in the eluant from the column, two major proteins (with molecular masses of 60 and 43 kDa) were detected by SDS-polyacrylamide gel electrophoresis. Based on the amino acid sequence analysis of these proteins, 60- and 43-kDa mizoribine-binding proteins were identified with HSP60 and cytosolic actin, respectively. A considerable amount of actin was also eluted from the affinity column by nucleotides, but a very low quantity of HSP60 was eluted under the same conditions. On the other hand, HSP60 was eluted as a major protein in the eluant that was eluted preferentially, with nucleotide followed by mizoribine. Actin was also detected in the eluant, but the quantity of the protein was very low. These results indicated that HSP60 has high affinity to mizoribine, and the interaction was also observed on surface plasmon resonance analysis. Although HSP60 or GroE facilitated refolding of citrate synthase in vitro, mizoribine interfered with the chaperone activity of HSP60. On different types of mizoribine affinity columns, HSP60 or actin recognized the NH(2) group of mizoribine, and this group may be a functional group of the agent.
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PMID:Mammalian HSP60 is a major target for an immunosuppressant mizoribine. 1057 97

The fiber type-specific expression of skeletal muscle GLUT4 and the effect of 2 weeks of low-intensity training were investigated in 8 young untrained male subjects. Single muscle fibers were dissected from a vastus lateralis biopsy sample. Based on myosin heavy chain (MHC) expression, fibers were pooled into 3 groups (MHC I, MHC IIA, and MHC IIX), and the GLUT4 content of 15-40 pooled fibers was determined using SDS-PAGE and immunological detection. The GLUT4 content in pooled muscle fibers expressing MHC I was approximately 20% higher (P < 0.05) than that in muscle fibers expressing MHC IIA or MHC IIX. No difference in GLUT4 could be detected between fibers expressing MHC IIA or MHC IIX. Two weeks of exercise training increased (P < 0.05) the peak power output of the knee extensors by 13%, the maximal activities of citrate synthase and 3-hydroxyacyl-CoA dehydrogenase by 21 and 18%, respectively, and the GLUT4 protein content by 26% in a muscle homogenate. Furthermore, a 23% increase (P < 0.05) in GLUT4 was seen in fibers expressing the MHC I isoform after exercise training for 2 weeks. No change was seen in fibers expressing MHC IIA or MHC IIX. In conclusion, our data directly demonstrate that GLUT4 is expressed in a fiber type-specific manner in human skeletal muscle, although fiber type differences are relatively small. In addition, low-intensity exercise training recruiting primarily fibers expressing MHC I increased GLUT4 content in these fibers but not in fibers expressing MHC IIA or MHC IIX, indicating that GLUT4 protein content is related more to activity level of the fiber than to its fiber type, which is defined by expression of contractile protein.
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PMID:Fiber type-specific expression of GLUT4 in human skeletal muscle: influence of exercise training. 1090 63


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