Phospholipase D hydrolyzes short-chain analogs of phosphatidylcholine in the absence of detergent.
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 [3H]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.
Davis
L L
LL
MCP Hahnemann School of Medicine, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19129, USA.
Maglio
J J
JJ
Horwitz
J
J
D000818:Animals; D001921:Brain / Q000201:enzymology; D000440:Butanols / Q000378:metabolism; D003902:Detergents; D006868:Hydrolysis; D008861:Microsomes / Q000201:enzymology; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats
C111346:1,2-dioctanoyl-sn-glycero-3-phosphocholine; D000440:Butanols; D003902:Detergents; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D010739:Phospholipase D