35] Phosphatidylserine synthase from yeast

Abstract

This chapter describes the purification, reconstitution, and properties of the phosphatidylserine synthase. Phosphatidylserine synthase catalyzes the incorporation of serine into phosphatidylserine. The enzyme plays an important role in the regulation of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae . Phosphatidylserine synthase activity is associated with the mitochondrial and microsomal fractions of S. cerevisiae . Phosphatidylserine synthase expression is regulated by inositol and in concert with serine, ethanolamine, and choline. Microsomal-associated phosphatidylserine synthase has been purified to near homogeneity using CDPdiacylglycerol-sepharose affinity chromatography. CDPdiacylglycerol is prepared from phosphatidic acid and CMPmorpholidate by the method of Agranoff and Suomi with the modifications of Carman and Fischl. The procedure for the electroblotting of phosphatidylserine synthase is similar to that used for the electroblotting of phosphatidylinositol synthase. The presence of CDPdiacylglycerol and cofactors in the renaturation buffer stabilizes phosphatidylserine synthase activity. The nitrocellulose paper is used for enzyme assays. The recovery of phosphatidylserine synthase activity is about 10%. This procedure allows the confirmation of the molecular weight of phosphatidylserine synthase. Pure phosphatidylserine synthase has been reconstituted into unilamellar phospholipid vesicles containing its substrate CDPdiacylglycerol. Pure phosphatidylserine synthase can be used to synthesize radiolabeled phosphatidylserine from CDPdiacylglycerol and labeled serine. The enzyme can be used to synthesize various fatty acyl derivatives of phosphatidylserine using the appropriate fatty acyl derivatives of CDPdiacylglycerol as substrate.