Determination of phospholipase activity of PAF acetylhydrolase.
This article presents a radiometric assay to determine the enzymatic activity of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2 and phospholipase A2 group 7A. The method is based on the release of radioactively labeled acetate from sn-2-labeled PAF and separation of substrate and product using reversed-phase column chromatography on octadecyl silica gel cartridges. The assay is fast, convenient, reproducible, sensitive, and inexpensive. The instrumentation required includes standard laboratory equipment and a liquid scintillation counter. The assay is also useful to determine the activity of intracellular PAF-AH (PAF-AH II), provided that a few modifications are included. The enzymatic activity determined using PAF as the substrate is a direct indication of the ability of plasma samples, purified preparations, and cellular and tissue lysates to hydrolyze short- and medium-chain phospholipids that may or may not harbor oxidized functionalities. In addition, the assay can be used to test the suitability of other phospholipids, including species containing oxidized, long-chain sn-2 fatty acyl groups, as PAF-AH substrates. This versatile assay can be used to accurately determine PAF-AH activity in biological samples and preliminarily assess affinity and efficiency of the hydrolysis of potential substrates present in complex mixtures.
Huntsman Cancer Institute and Department of Internal Medicine, University of Utah, Salt Lake City, UT 84112, USA. diana.stafforini@hci.utah.edu
Diana M
DM
Stafforini
Thomas M
TM
McIntyre
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase / Q000097:blood / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D010740:Phospholipases / Q000097:blood / Q000378:metabolism*
D010740:Phospholipases; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase