Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry Vol. 262; no. 30; pp. 14563 - 14570
• Main Authors: Kelley, M J, Carman, G M
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.10.1987; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Affinity; Chromatography, Ion Exchange; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Molecular Weight; Nucleotidyltransferases - immunology; Nucleotidyltransferases - isolation & purification; Nucleotidyltransferases - radiation effects; Octoxynol; phosphatidate cytidylyltransferase; Polyethylene Glycols - pharmacology; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Temperature; Transferases; Fungi; Phosphatidate cytidylyltransferase; Yeast; Antibody; Affinity chromatography; Enzyme; Preparation; Ascomycetes; Catalyst activity; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)47833-7

The membrane-associated phospholipid biosynthetic enzyme CDP-diacylglycerol synthase (CTP:phosphatidate cytidylyltransferase, EC 2.7.7.41) was purified 2,300-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of mitochondrial membranes, CDP-diacylglycerol-Sepharose affinity chromatography, and hydroxylapatite chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radiation inactivation of mitochondrial associated and purified CDP-diacylglycerol synthase suggested that the molecular weight of the native enzyme was 114,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme preparation yielded two subunits with molecular weights of 56,000 and 54,000. Antibodies prepared against the purified enzyme immunoprecipitated CDP-diacylglycerol synthase activity and subunits. CDP-diacylglycerol synthase activity was dependent on magnesium ions and Triton X-100 at pH 6.5. Thio-reactive agents inhibited activity. The activation energy for the reaction was 9 kcal/mol, and the enzyme was thermally labile above 30 degrees C. The Km values for CTP and phosphatidate were 1 and 0.5 mM, respectively, and the Vmax was 4,700 nmol/min/mg. Results of kinetic and isotopic exchange reactions suggested that the enzyme catalyzes a sequential Bi Bi reaction mechanism.