Phosphatidate phosphydrolase (PAP) exists in two forms in mammalian cells. PAP-1 is Mg2+-dependent, cytosolic, and inhibited by N-ethylmaleimide. PAP-2 is membrane-bound, Mg2+-independent, and insensitive to N-ethyl maleimide.

50 mM Tris-HCl, pH 7.1; 10 mM b-mercaptoethanol, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM Triton X-100; 100 mM phosphatidic acid (14C-labeled).

Substrate in the form of mixed micelles detergent/phospholipid (10:1), Radioactive phosphatidate usually purchased from NEN, but they may have discontinued production. ARC used to serve it as well.

After the reaction, [14C]diacylglycerol is separated from [14C]phosphatidate by thin-layer chromatography, utilizing n-hexane/diethyl ether/acetic acid (70:30:1) as a solvent system.