Purification and some properties of the citrate synthase from a marine Pseudomonas.
Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.
D000227:Adenine Nucleotides / Q000494:pharmacology; D002950:Citrate (si)-Synthase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification; D011189:Potassium Chloride / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D012623:Seawater; D014871:Water Microbiology
D000227:Adenine Nucleotides; D007527:Isoenzymes; D009243:NAD; D011189:Potassium Chloride; D004228:Dithionitrobenzoic Acid; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases