Abstract
This chapter describes the purification process and properties of phosphatidate phosphatase from yeast. [γ-32P]Phosphatidate is synthesized enzymatically from [γ-32P]adenosine triphosphate (ATP) and diacylglyccrol using Escherichia coli diacylglycerol kinase. [2-3H]Phosphatidate is prepared from [2-3H]glycerol 3-phosphate and palmitoyl-CoA using yeast microsomal-associated glycerol-3-phosphate acyltransferase. Labeled phosphatidate is purified by thin-layer chromatography using the solvent system chloroform/methanol/water. Phosphatidate phosphatase activity is routinely measured by following the release of water-soluble [32P]Pi from 0.5 mM [γ-32P]phosphatidate [1000-2000 counts per minute (cpm)/nmol] in 50 mM Tris-maleate buffer (pH 7.0) containing 5 nM Triton X-100, 10 mM 2-mercaptoethanol, 2 mM MgCl2, and enzyme protein in a total volume of 0.1 ml at 30°. The reaction is terminated by the addition of 0.5 ml of 0.1 N HCl in methanol. Chloroform (1 ml) and I M MgCl2 (1 ml) are added, the system is mixed, and the phases are separated by a 2-min centrifugation at 100 g. Four milliliters of Liquiscint is added to a 0.5-ml aliquot of the aqueous phase, and radioactivity is determined by scintillation counting. Phosphatidate phosphatase activity is measured by following the formation of [2-3H]diacylglycerol from [2-3H]phosphatidate (1000 cpm/nmol).