FLUORESCENCE METHOD FOR STUDY OF CABBAGE PHOSPHOLIPASE-D ACTIVITY

Phospholipase D (Lecithinase D; phosphatidylcholine phosphatidohydrola se, EC 3.1.4.4; PLase D), an important enzyme in intracellular signali ng, catalyzes the hydrolysis of phospholipids to produce free polar he ad groups and phosphatidic acid, a potent cytokine in many cells. A fl uorescent assay for PLase D activity has been developed utilizing ino- 1-naphthalenesulfonyl-phosphatidylethanolamine (DNS-PE) as a fluoresce nt reporter molecule. The validity of this assay was determined by mea suring loss of [C-14]phosphatidylcholine (PC) and formation of [C-14]p hosphatidic acid by high performance liquid chromatography. Using the fluorescent assay, a partially purified cabbage PLase D preparation hy drolyzed 0.67 nmol PC min(-1) mu g protein(-1) protein corresponding t o changes in polarization of fluorescence of DNS-PE of 6.6% mu g prote in min(-1) ml(-1). Optimum activity for the assay was observed in the presence of 32 mu M phosphatidylcholine, 3.2 mu M DNS-PE, 0.002-0.004% sodium dodecyl sulfate and cabbage PLase D (1-2 mu g protein ml(-1)) in 60 mM (4-morpholino)-ethanesulfonic acid buffer (pH 5.5) when 6 mM CaCl2 was used to initiate the reaction. The use of inhibitors such as tricyclodecan-9-yl-xanthogenate, which blocked phospholipase C activi ty, and ZnCl2, which blocked PLase D activity, may be especially usefu l in defining potential contributions of relevant enzymes to the chang es in polarization of fluorescence. This fluorescence assay provides a convenient means for further study of PLase D activity in purified pr eparations of the enzyme.