Purification and characterization of CDP-diacylglycerol synthase from Saccharomyces cerevisiae.
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.
Kelley
M J
MJ
Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick 08903.
Carman
G M
GM
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000276:immunology / Q000302:isolation & purification* / Q000528:radiation effects; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013696:Temperature
D011092:Polyethylene Glycols; D017830:Octoxynol; D009713:Nucleotidyltransferases; C022348:phosphatidate cytidylyltransferase