Group-Specific Assays That Distinguish between the Four Major Types of Mammalian Phospholipase A2

Abstract

Phospholipase A2(PLA2) constitutes a diverse superfamily of enzymes which catalyze the deacylation of phospholipids. At least four types of PLA2are potentially involved in arachidonic acid release in cells and tissues. Since all of them catalyze the same enzymatic reaction, it is difficult to distinguish between them in mixtures of enzymes normally present in biological samples. Utilizing specific properties of each PLA2, we have designed distinct assay procedures which selectively and sensitively detect each type: Group VI Ca2+-independent PLA2, Group IV cytosolic Ca2+-dependent PLA2, Groups V and IIA secreted PLA2s. Each specific assay procedure is selective for a particular PLA2type by at least fourfold and as high as four orders of magnitude relative to the other three enzymes. All assays can detect PLA2activity with as low as subnanogram quantities of enzyme. Importantly, these assays are able to differentiate and quantitate the biochemically and structurally related enzymes, Group IIA and V sPLA2s in crude biological samples. Employing this system, we have found that iPLA2is the dominant PLA2in rat brain, and cPLA2is the most abundant PLA2in P388D1macrophages and human amnionic WISH cells.