Disease relevance of CS

• 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] [1].
• RESULTS: Relative to the control group, the exercise group demonstrated typical exercise adaptations of increased ventricular weight/body weight ratio, enhanced skeletal muscle citrate synthase activity and higher concentrations of [3H]ouabain binding sites in both skeletal and cardiac tissue (P<0.05) [2].
• Sequence and linkage analysis of the Coxiella burnetii citrate synthase-encoding gene [3].
• The effects of normothermia and delayed hypothermia on the levels of N-acetylaspartate (NAA), reduced glutathione (GSH) and the activities of mitochondrial complex I, II-III, IV and citrate synthase were measured in brain homogenates obtained from anaesthetized neonatal pigs following transient in vivo hypoxia-ischaemia [4].

High impact information on CS

• The sequence of 437 amino acid residues of porcine heart citrate synthase [citrate oxaloacetate-lyase (pro-3S-CH2COO leads to acetyl-CoA), EC 4. 1. 3. 7] has been determined by the alignment of fragments generated by cleavage with cyanogen bromide and with trypsin [5].
• This new analogue is not as good (by an order of magnitude) an inhibitor of citrate synthase [citrate oxaloacetatelyase (pro-3S-CH2-COO-vectoracetyl-CoA); EC 4.1.3.7] nor is it bound as well oleoyl-CoA [6].
• Inhibition of citrate synthase by oleoyl-CoA: a regulatory phenomenon [6].
• Since the only difference between these two compounds is substitution of 1,N6-ethenoadenine for the adenine of CoA, the difference in inhibition and binding implies a specific interaction between the adenine moiety of oleoyl-CoA and citrate synthase [6].
• The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them [7].

Chemical compound and disease context of CS

• 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)[1]
• CS was expressed in E. coli by using an arabinose regulated T7 promotor [8].
• We report properties of five active site mutants of Escherichia coli citrate synthase, in which histidine 264, aspartate 362, and phenylalanine 383 were replaced by alanines, and arginines 387 and 407 by leucines [9].
• Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively [10].
• Therefore, the surface profiles of citrate synthase enzymes from yeast, pig, rat, tomato and Escherichia coli were investigated with immunological methods using monoclonal antibody families generated against either pig citrate synthase (alpha-PCS) or yeast citrate synthase-2 (alpha-YCS-2) [11].

Biological context of CS

• Citrate synthase (CS) is a key enzyme of the Krebs tricarboxylic acid cycle [12].
• The CS gene was therefore assigned to the p12-p13 bands of chromosome 5 in pigs [12].
• 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 [1].
• 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 [1].
• Isolation, nucleotide sequence, and expression of a cDNA encoding pig citrate synthase [1].

Anatomical context of CS

• Muscles exhibiting increased CS activity, however, were located primarily in the forelimb; ankle and knee extensor and respiratory muscles were unchanged with training [13].
• The conformation of the enzyme is essentially identical with that of a previously determined "open" form of pig heart muscle citrate synthase which crystallizes in a different space group, with one monomer in the asymmetric unit, from either phosphate or citrate solution [14].
• Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function [15].
• Major differences were found between the hexameric citrate synthase originating from E. coli compared with those dimeric forms prepared from eukaryotic cells [11].
• The enzyme, CS2, the sequence of which had been previously determined from its DNA, behaved differently from CS1 in its purification, kinetics, stability, and binding to the inner surface of mitochondrial inner membranes [16].

Associations of CS with chemical compounds

• 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 [1].
• Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested [7].
• Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 mug/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro [7].
• The co-treatment of FB(1) (CS inhibitor) with SPT inhibitors or the GlcCer synthase inhibitor had no effect on the FB(1)-induced reduction in pERK2 phosphorylation, indicating that FB(1)-mediated changes in phosphorylation of pERK2 was independent of increases in free sphinganine or its metabolites or a reduction in ceramide [17].
• Lower CS and HAD activities were observed in the halothane sensitive pigs compared with the other pigs [18].

Other interactions of CS

• Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase [19].
• The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected [19].
• The mitochondrial enzymes citrate synthase, malate dehydrogenase, and aspartate aminotransferase were purified to homogeneity from porcine hearts by use of Bio-Rex 70, carboxymethylcellulose CM32, and Affi-Gel blue chromatography [20].
• Our results support the hypothesis of cluster formation between the glycolytic enzymes aldolase, glyceraldehydephosphate dehydrogenase, and triosephosphate isomerase, and between the cycle enzymes fumarase, malate dehydrogenase, and citrate synthase [21].
• MATERIALS AND METHODS: 60Co gamma-irradiation of three proteins at -78 degrees C: lysozyme, citrate synthase and alpha-lactalbumin in their native state, with or without bound substrate, or denatured (random coil in urea/acid-denatured state) [22].

Analytical, diagnostic and therapeutic context of CS

• A 1.4 kb porcine CS cDNA probe was used to chromosomally localize the CS gene in pigs by in situ hybridization [12].
• The presence of the CS-like, CS-A, GH-variant and, most downstream, CS-B gene was confirmed by DNA blotting and sequence analysis [23].
• After these two steps of anion-exchange chromatography a final size-exclusion chromatography step on a Superdex 75-pg column yields CS with a purity over 99% [8].
• A CS cDNA probe was prepared from a rabbit heart cDNA library by the polymerase chain reaction using synthetic oligonucleotide primers based on the published sequence of the porcine gene [24].
• Using purified IgG, competitive enzyme-linked immunoassays and assays of citrate synthase activity indicate the presence of antibodies to a number of antigenic sites on the enzyme, only some of which are essential for catalytic activity [25].

References