Preparation of thioester substrates and development of continuous spectrophotometric assays for phospholipase A1 and monoacylglycerol lipase

Abstract

Current assays of phospholipase A1 (EC 3.1.1.32) and monoacylglycerol lipase (EC 3.1.1.23) activities in tissues are discontinuous, laborious, and expensive. Some spectrophotometric substrates were synthesized to alleviate this problem. Thioester analogs of phosphatidylcholine and phosphatidylethanolamine. rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-3-phosphoethanolamine-1-mercapto-2,3-propanediol, were synthesized from the diacylglycerol analog, rac-1,2-S,O–didecanoyl-1-mercapto-2,3-propanediol. The latter was prepared from triacylmercaptoglycerol by lipolysis and separation by chromatography on silica gel. Monoacylglycerol thioester analogs, 1-S-hexadecanoyl-1-mercapto-2,3-propanediol and 1-S-decanoyl-1-mercapto-2,3-propanediol, were synthesized by selective acylation of mercaptoglycerol. All of the substrates were hydrolyzed by Rhizopus delemar lipase to release sulfhydryl groups reactive towards 4,4'-dithiobispyridine. The hydrolysis could be followed continuously in a spectrophotomer with 0.1 absorbance unit corresponding to 5 nmol product. The structure and isomeric purity of the phospholipid analogs were verified by their behavior on thin-layer chromatography, elemental analyses, infrared spectra, and by the specificity of the colorimetric reaction with lipolytic enzymes. Whereas phospholipase A1 activity hydrolyzed both phospholipid analogs to release the theoretical amount of free thiol, neither phospolipases C nor A2 promoted thio release. The pH optimum, heat stability, and Ca2+ ion dependency were determined for the hydrolysis of each substrate by R. delemar lipase. The results indicate that the phospholipase A1 and monoacylglycerol lipase activities in R. delemar lipase are due to separate enzymes, and that these enzyme specific assays will be of general utility for enzyme characterization and purification studies. These substrates are useful for sensitive, convenient, and specific spectrophotometric assays for phospholipase A1 and monoacylglycerol lipase over the pH range 3 to 8.