A facile method for direct determination of phospholipase A2 activity in intact cells

The fluorescent phospholipid analogue 1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3- diazole)aminocaproylphosphatidylcholine (C6-NBD-PC), which incorporates into cell membranes, is employed as a substrate for phospholipase A2 (PLA2) in intact cells. The interaction of this substrate with the cells produces only one fluorescent product; the fatty acid C6-NBD-FA, which does not incorporate into other lipids, and is not further metabolized. The product, a hydrophilic fatty acid, is separated from the substrate by aqueous: organic solvent phase separation. Using this method, the fatty acid produced is fully recovered and its amount, as measured by its fluorescence intensity, is a direct measure of the cell membrane PLA2 activity.