Abstract
Phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase) catalyzes the conversion of phosphatidate to diacylglycerol. In the yeast, Saccharomyces cerevisiae (S. cerevisiae), phosphatidate phosphatase is associated with the membrane and cytosolic fractions of the cell. The enzyme plays an important role in the biosynthesis of phospholipids and triacylglycerols in S. cerevisiae. Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form of phosphatidate phosphatase has revealed the existence of a 45-kDa form of the enzyme. This immunoblot analysis has also revealed that the 91-kDa enzyme is a proteolysis product of a 104-kDa enzyme. The mitochondrial fraction contains the 45-kDa enzyme, whereas the microsomal fraction contains the 45- and 104-kDa enzymes. 7 The 45-kDa phosphatidate phosphatase is induced in yeast cells by inositol supplementation, whereas the 104-kDa enzyme is not affected by inositol. Both forms of the enzyme are induced when cells enter the stationary phase of growth. The phosphatidate phosphatase 45-kDa enzyme has been purified from yeast mitochondria by a procedure similar to that used to purify the phosphatidate phosphatase 91-kDa enzyme from total membranes. The chapter describes the purification and properties of the 45-kDa form of the enzyme.