Abstract
Phosphatidylinositol synthase catalyzes the incorporation of inositol into phosphatidylinositol. The enzyme and its product phosphatidylinositol are essential to the growth of the yeast Saccharomyces cerevisiae (S. cerevisiae). Phosphatidylinositol synthase activity is associated with the mitochondrial, microsomal, and plasma membrane fractions of S. cerevisiae. Microsome-associated phosphatidylinositol synthase has been purified to near homogeneity, primarily by CDPdiacylglycerol-sepharose affinity chromatography. This chapter describes the purification, reconstitution, and properties of the enzyme. CDPdiacylglycerol is prepared from phosphatidic acid and CMPmorpholidate by the method of Agranoff and Suomi with the modifications of Carman and Fischl. Phosphatidic acid is prepared from soybean lecithin by reaction with cabbage phospholipase D. The chapter discusses the preparation of CDPdiacylglycerol-sepharose. Electroblotting of phosphatidylinositol synthase activity is described. Pure phosphatidylinositol synthase has been reconstituted into unilamellar phospholipid vesicles containing its substrate CDPdiacylglycerol. Pure phosphatidylinositol synthase can be used to synthesize radiolabeled phosphatidylinositol from CDPdiacylglycerol and labeled inositol.