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Phospholipase D hydrolyzes short‐chain analogs of phosphatidylcholine in the absence of detergent
Author(s) -
Davis Leslee L.,
Maglio Jeffrey J.,
Horwitz Joel
Publication year - 1998
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-998-0199-5
Subject(s) - phosphatidylcholine , lipidology , clinical chemistry , chemistry , phospholipase , hydrolysis , biochemistry , phospholipase c , phospholipase d , chromatography , enzyme , phospholipid , membrane
Phospholipase D is an important enzyme in signal transduction in neuronal tissue. A variety of assays have been used to measure phospholipase D activity in vitro . The most typical measure of phospholipase D activity is the production of phosphatidylethanol in the presence of ethanol. Phosphatidylethanol is a product of transphosphatidylation activity that is considered a unique property of phospholipase D. To support transphosphatidylation activity, high concentrations of ethanol may be required. Furthermore, most assays in the literature utilize a detergent. These extreme conditions, detergent and ethanol, may alter phospholipase D and hinder the study of its regulation. In this manuscript we describe an assay that eliminates these potentially confounding conditions. It utilizes high specific activity [ 3 H]butanol as a nucleophilic receptor. This eliminates the need for high concentrations of alcohol. The substrate is an analog of phosphatidylcholine that contains short‐chain fatty acids, 1,2‐dioctanoyl‐ sn ‐glycero‐3‐phosphocholine. Phospholipase D readily hydrolyzes this substrate in the absence of detergent. This novel assay should be useful in the further characterization of phospholipase D.