Assaying Phospholipase A2 Activity
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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.
