Abstract
The chapter presents a study on macrophage phospholipase A2 specific for sn-2-arachidonic acid. Because most arachidonic acid is esterified at the sn-2 position of phospholipid, phospholipase A2 is thought to play a central role in providing arachidonic acid for subsequent metabolism. In addition, phospholipase A2-mediated hydrolysis of arachidonic acid from the ether-linked membrane phospholipid, 1-O-alkyl-2-arachidonoylglycerophosphocholine (–GPC), is the first step in the production of the potent phospholipid mediator platelet-activating factor (PAF) in inflammatory cells. The lyso-PAF formed is then acetylated to PAF. Compared to diacyl-GPC, the 1-O-alkyl-linked species is enriched in arachidonic acid in neutrophils and macrophages. Consequently, the eicosanoids and PAF can be coordinately produced from a common phospholipid precursor through the initial action of a phospholipase A2. The chapter describes the assay of this arachidonoylspecific phospholipase A2 from the macrophage cell line, RAW 264.7 and the preparation of the 1-O-hexadecyl-2-[3H]arachidonoyl-GPC substrate. The RAW 264.7 macrophage cell line, originally established from a murine tumor induced by Abelson's leukemia virus, secretes lysozyme, is phagocytic, and has receptors for complement and immunoglobulin. In addition, the RAW 264.7 cells release arachidonic acid in response to zymosan and endotoxin.
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