Assay of Phospholipase D Activity in Cell-Free Systems

Phospholipase D (PLD) enzymes are present in all animal and plant species and have been linked to many critical cellular processes, including proliferation, differentiation, motility, and secretion. The functional significance of PLD derives from its generation of phosphatidic acid, which has both direct signaling properties via activation of numerous kinases, phosphatases, phopspholipases, and other enzymes, as well as via its conversion to diglycerides, the endogenous activators of protein kinase C. The two mammalian PLD isoforms, PLD1 and PLD2, are peripheral membrane proteins that exhibit important physical and functional interactions with the actin cytoskeleton. We outline a cell-free system for the characterization of mammalian PLDs and their activation by physiologic stimuli or pharmacologic agonists for guanine triphosphate-binding proteins. This assay system is used to illustrate the interactions of PLD1 with specific membrane domains and their associated filamentous and monomeric actin components.