Determination of phospholipase activity of PAF acetylhydrolase

Abstract

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.