Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae.
Membrane-associated cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC2.7.8.8.) and CDP-diacylglycerol:myo-inositol phosphatidyltransferase (phosphatidylinositol synthase, EC 2.7.8.11) were solubilized from the microsomal fraction of Saccharomyces cerevisiae. A variety of detergents were examined for their ability to release phosphatidylserine synthase and phosphatidylinositol synthase activities from the microsome fraction. Both enzymes were solubilized from the microsome fraction with Renex 690 in yield over 80% with increase to specific activity of 1.6-fold. Both solubilized enzymatic activities were dependent on manganese ions and Triton X-100 for maximum activity. The pH optimum for each reaction was 8.0. The apparent Km values for CDP-diacylglycerol and serine for the phosphatidylserine synthase reaction were 0.1 and 0.25 mM, respectively. The apparent Km values for CDP-diacylglycerol and inositol for the phosphatidylinositol synthase reaction were 70 microM and 0.1 mM, respectively. Thioreactive agents inhibited both enzymatic activities. Both solubilized enzymatic activities were thermally inactivated at temperatures above 30 degrees C.
D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D003568:Cytidine Monophosphate / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility; D013696:Temperature; D017855:Transferases (Other Substituted Phosphate Groups)
D002413:Cations, Divalent; D003902:Detergents; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D010717:CDPdiacylglycerol-Serine O-Phosphatidyltransferase; D003568:Cytidine Monophosphate