PHOSPHOLIPASE-D HYDROLYZES SHORT-CHAIN ANALOGS OF PHOSPHATIDYLCHOLINEIN THE ABSENCE OF DETERGENT

Phospholipase D is an important enzyme in signal transduction in neuro nal tissue. A variety of assays have been used to measure phospholipas e D activity in vitro. The most typical measure of phospholipase D act ivity is the production of phosphatidylethanol in the presence of etha nol. Phosphatidylethanol is a product of transphosphatidylation activi ty that is considered a unique property of phospholipase D. To support transphosphatidylation activity, high concentrations of ethanol may b e required. Furthermore, most assays in the literature utilize a deter gent. These extreme conditions, detergent and ethanol, may alter phosp holipase D and hinder the study of its regulation. In this manuscript we describe an assay that eliminates these potentially confounding con ditions. It utilizes high specific activity [H-3]butanol as a nucleoph ilic receptor. This eliminates the need for high concentrations of alc ohol. The substrate is an analog of phosphatidylcholine that contains short-chain fatty acids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine. P hospholipase D readily hydrolyzes this substrate in the absence of det ergent. This novel assay should be useful in the further characterizat ion of phospholipase D.