DIDECANOYL PHOSPHATIDYLCHOLINE IS A SUPERIOR SUBSTRATE FOR ASSAYING MAMMALIAN PHOSPHOLIPASE-D

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techni ques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extrac ted and partially purified on one column before easy detection of acti vity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5'-[ga mma-thio]-triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ri bosylation factor (Arf)]. In this paper we report that the use of dide canoyl phosphatidylcholine (C-10-PC) in mammalian PLD assays considera bly increases the detection limit. C-10-PC was compared with the commo nly used dipalmitoyl phosphatidylcholine (C-16-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and p ig brain, and from placental cytosol. C-10-PC was superior to C-16-PC by a factor of 2-28 depending on assay conditions and tissue, and it a llowed the detection of GTP[S]-and Arf-stimulated PLD activity without addition of phosphatidylinositol 4,5-bisphosphate.