Hydrolysis of N-acylated glycerophospholipids by phospholipases A2 and D: a method of identification and analysis.
We have previously identified N-acylethanolamine phospholipids in infarcted dog heart and in normal fish brain by chemical and enzymatic degradation. We now report that hydrolysis with phospholipase D from Streptomyces chromofuscus removes N-acylethanolamine from N-acylethanolamine phospholipids and lyso N-acylethanolamine phospholipids, or N-acylserine from lyso N-acylserine phospholipids. At acidic pH, a phosphatase present in the phospholipase D preparation further hydrolyzes the resulting phosphatidic acid (PA) or lyso-PA to diacyl- or monoacylglycerol. Because N-acylserine phospholipids are a poor substrate for the phospholipase D, pretreatment with phospholipase A2 (Trimeresurus flavoviridis venom) is used to remove the 2-O-acyl group. Thus, both types of N-acylated phospholipids can be analyzed by consecutive phospholipase A2 and phospholipase D treatment. Reaction products, i.e., free fatty acids, monoacylglycerols and N-acylethanolamine or N-acylserine, are separable by thin-layer chromatography. Both N-acyl components can be further characterized by conversion to the t-butyldimethylsilyl derivatives. The method was used to identify and analyze the N-acylserine phospholipids of bovine brain.