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.
Department of Biochemistry, Hebrew University, Hadassah School of Medicine, Jerusalem, Israel.
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Dagan
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Yedgar
D000818:Animals; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D005227:Fatty Acids / Q000032:analysis; D008055:Lipids / Q000032:analysis; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D051381:Rats; D013050:Spectrometry, Fluorescence
D005227:Fatty Acids; D008055:Lipids; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2