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Download Citrate synthase
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Storage temperature Citrate synthase should be stored at 4 °C or and will remain stable up to 3 years if stored as specified. Citrate synthase (EC 2.3.3.1), Escherichia coli Temperature and pH optimum The optimum pH and temperature are 8.0 and 25 °C, respectively. Specific activity Catalogue number: AE00041, 2500 U (148 mg) 16.9 U/mg, 186 U/ml. Unit definition Description Citrate synthase (E.C. 2.3.3.1) is purified from a recombinant E. coli strain. The enzyme exists in nearly all living cells and stands as a pace-making enzyme in the first step of the Krebs Cycle. Citrate synthase is localized within eukaryotic cells in the mitochondrial matrix, but is encoded by nuclear DNA rather than mitochondrial. It is synthesized using cytoplasmic ribosomes, then transported into the mitochondrial matrix. Citrate synthase is commonly used as a quantitative enzyme marker for the presence of intact mitochondria. Citrate synthase catalyses the condensation reaction of acetyl-CoA and oxaloacetate producing citrate. Oxaloacetate will be regenerated after the completion of one round of the Krebs Cycle. Oxaloacetate is the first substrate to bind to the enzyme. This induces the enzyme to change its conformation, and creates a binding site for the acetyl-CoA. Only when this citroyl-CoA has formed will another conformational change cause thioester hydrolysis and release coenzyme A. This ensures that the energy released from the thioester bond cleavage will drive the condensation. The enzyme is provided in 3.2 M ammonium sulphate. Swirl the enzyme mix immediately prior to use. Purity Citrate synthase has been determined to be >95% pure, according to SDS polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Blue staining (Figure 1). kDa 96 66 48 40 32 26 18.5 M 1 Figure 1. SDS-PAGE analysis of E. coli citrate synthase. Electrophoresis was performed using a 12% polyacrylamide gel. Lane M, molecular weight marker; Lane 1, purified citrate synthase (48 kDa). One unit is defined as the amount of enzyme required to produce 1 mol of citric acid from oxaloacetic acid and acetyl-CoA measured at 232 nm, in a reaction mixture containing 95 mM Tris/HCl buffer (pH 8.0), 0.16 mM oxaloacetic acid and 0.2 mM acetyl-CoA. Substrate specificity Under the reaction conditions specified the enzyme might present a minor NADH oxidase activity. Enquire [email protected] to obtain any citrate synthase mutant derivative. Revised 12/12 Certificate of Analysis Test Criteria Result Protein purity Purity in line with the stated value Meets specification Protein concentration Concentration in line with the stated value Meets specification Catalytic activity Activity in line with the stated value Meets specification Blank assay variability Absorbance values with less than 10% of variability Meets specification Approved by: José Prates Senior Manager, Quality Systems Please enquire [email protected] to obtain any additional information. Bulk quantities of this product are available on request. Estrada do Paço do Lumiar, Campus do Lumiar - Edifício E, R/C 1649-038 Lisboa, Portugal Tel.:+351.213643514 Fax: +351.217151168 www.nzytech.com