Assaying Phospholipase A2 Activity

Mammalian cells contain many structurally and functionally diverse phospholipases A₂ (PLA₂ ) that catalyze the hydrolysis of sn-2 fatty acid from membrane phospholipid. Assays are described for measuring the activity of Group IVA cytosolic PLA₂ α (cPLAα) and for secreted PLA₂ s (sPLA₂ ) that are suitable for purified enzymes and for measuring activity in crude cell lysates and culture medium. The assay for cPLA₂ α involves measuring the calcium-dependent release of radiolabeled sn-2 arachidonic acid from small unilamellar vesicles of phosphatidylcholine. Methods are described for distinguishing cPLA₂ α activity in cell lysates from other PLA₂ s. sPLA₂ activity is monitored using a fluorimetric assay that measures the continuous calcium-dependent formation of albumin-bound pyrene fatty acid from the sn-2 position of phosphatidylglycerol.