Measurement of Phospholipase D Activity

The importance of diacylglycerol (DAG) as a second messenger for calcium-mobilizing hormones, neurotransmitters, and cytokines was underscored by the discovery of an effector enzyme, protein kinase C, which was also a cellular receptor for phorbol ester tumor promoters (reviewed in ref. 1 ). However, with continued study it became apparent that hydrolysis of the polyphosphoinositides by phosphoinositide-specific phospholipase C was not the only pathway through which DAG could be generated (reviewed in ref. 2 ). In addition to phospholipases C that utilize other phospholipids as substrates (e.g., phosphatidylcholine-specific phospholipase C), a mechanism for DAG production involving the combined action of phospholipase D (PLD) and phosphatidate phosphohydrolase has been demonstrated (reviewed in ref. 3 ). Thus, phospholipid hydrolysis by PLD yields phosphatidic acid (PA), which can be dephosphorylated by phosphatidate phosphohydrolase to produce DAG. Furthermore, it appears that, whereas hormone-induced phosphoinositide hydrolysis by phospholipase C is an important pathway of initial DAG formation, sustained DAG generation often arises through this second PLD mechanism (reviewed in ref. 1 ).