Measurement of Phospholipase D Activity

Abstract

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).