Phosphatidylinositol synthase catalyzes the formation of phosphatidylinositol from CDP-diacylglycerol and inositol. Phosphatidylinositol is a major eukaryotic membrane component important to both normal and abnormal cell growth and division. The regulation of phosphatidylinositol synthase will be studied by biochemical and recombinant DNA methods. A purified preparation of phosphatidylinositol synthase isolated from yeast will be used to study the enzymological, physical and chemical properties of the enzyme. Kinetic studies will be carried out in detergent-phospholipid substrate systems and liposome systems in the absence of detergent. Highly specific antibody to phosphatidylinositol synthase will be prepared and used for biochemical and cloning studies. The structural gene for phosphatidylinositol synthase will be cloned in yeast by transformation with a YEp13 hybrid plasmid clone bank. Acquisition of a phosphatidylinositol synthase clone will allow the study of the regulation of phosphatidylinositol biosynthesis at the gene level as well as providing an overproduced source of the enzyme for biochemical studies. Phosphatidylserine synthase catalyzes the formation of phosphatidylserine from CDP-diacylglycerol and serine. The regulation of phosphatidylserine synthase must regulate the activity of phosphatidylinositol synthase since both enzymes must compete for the common substrate CDP-diacylglycerol. Phosphatidylserine synthase will be purified and studied by similar methods developed for the study of phosphatidylinositol synthase. The competition of phosphatidylinositol synthase and phosphatidylserine synthase for CDP-diacylglycerol will be studied in liposome systems. The results of these studies should shed light on the nature of eukaryotic membrane-bound enzymes and phospholipid biosynthesis. Results found with the yeast system should be relevant to higher eukaryotic organisms.
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