A rapid sensitive assay for phosphatidate phosphohydrolase.

For a purified preparation of the soluble form of phosphatidate phosphohydrolase (EC 3.1.3.4) from guinea pig cerebral cortex, I-O-alkyl-racglycerol 3-phosphate was found to be accepted as a substrate. This substrate analog was tritium-labeled in order to serve in a rapid sensitive assay for the enzyme, in which labeled I-alkyl glycerol is released. Heat denaturation and enzyme activity dependence on pH indicated that I-O-alkyl-rat-glycerol 3phosphate phosphohydrolase and phosphatidate phosphohydrolase activities in the preparation are attributable to the same enzyme. I-O-Alkyd-rat-glycerol 3phosphate was hydrolyzed with a V,, of 1.7 nmol min-’ mg-’ of protein and a K, of 270 PM.