A rapid assay for activity of phospholipase A2 using radioactive substrate.
A rapid method for the assay of phospholipase A2 has been developed using a radioactive substrate, L-alpha-dipalmitoyl-(2-[9,10(N)-3H]palmitoyl)-phosphatidylcholine. The substrate diluted with cold carrier (1 mM) is dissolved in 80% ethanol containing 25 mM sodium deoxycholate. The enzymatic reaction is performed in 1.0 ml 0.1 M glycine-NaOH buffer, pH 9.0, containing 2 mumol CaCl2, 10 micrograms bovine serum albumin, 2.5 mumol sodium deoxycholate, 0.01 unit (or less) phospholipase A2, and 40-100 nmol substrate. The enzymatic reaction is terminated by adding 0.2 ml 5% Triton X-100 solution containing 40 mumol EDTA. The product of the enzymatic reaction, radioactive palmitic acid, is extracted by 10 ml hexane containing 0.1% acetic acid in the presence of anhydrous sodium sulfate (0.5 g/ml). Activity of phospholipase A2 is directly determined from the radioactivity in the hexane extract. The present method achieves a quick separation of the radioactive product, [3H]palmitic acid, from the radioactive substrate, L-alpha-dipalmitoyl-(2-[3H]palmitoyl)-phosphatidylcholine, without the need of separation by TLC.
D000433:1-Propanol; D002855:Chromatography, Thin Layer; D003840:Deoxycholic Acid; D006586:Hexanes; D007553:Isotope Labeling; D017830:Octoxynol; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000302:isolation & purification; D010740:Phospholipases / Q000032:analysis*; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D011092:Polyethylene Glycols; D011663:Pulmonary Surfactants / Q000378:metabolism; D012824:Silicic Acid; D013379:Substrate Specificity; D014316:Tritium; D014867:Water
D006586:Hexanes; D010169:Palmitic Acids; D011663:Pulmonary Surfactants; D003840:Deoxycholic Acid; D014867:Water; D014316:Tritium; D012824:Silicic Acid; D019308:Palmitic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; D000433:1-Propanol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2