Cox J W, Horrocks L A
J Lipid Res. 1981 Mar;22(3):496-505.
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.
目前对组织中磷脂酶A1（EC 3.1.1.32）和单酰甘油脂肪酶（EC 3.1.1.23）活性的测定是不连续的、费力的且昂贵的。合成了一些分光光度法底物以缓解这一问题。从二酰甘油类似物rac-1,2-S,O-二癸酰基-1-巯基-2,3-丙二醇合成了磷脂酰胆碱和磷脂酰乙醇胺的硫酯类似物，即rac-1,2-S,O-二癸酰基-3-磷酰胆碱-1-巯基-2,3-丙二醇和rac-1,2-S,O-二癸酰基-3-磷酰乙醇胺-1-巯基-2,3-丙二醇。后者由三酰基巯基甘油通过脂解制备，并通过硅胶柱色谱分离。通过巯基甘油的选择性酰化合成了单酰甘油硫酯类似物，即1-S-十六酰基-1-巯基-2,3-丙二醇和1-S-癸酰基-1-巯基-2,3-丙二醇。所有底物均被德氏根霉脂肪酶水解，释放出对4,4'-二硫代二吡啶有反应性的巯基。水解过程可在分光光度计中连续监测，0.1个吸光度单位对应5 nmol产物。通过磷脂类似物在薄层色谱上的行为、元素分析、红外光谱以及与脂解酶的比色反应的特异性来验证其结构和异构体纯度。虽然磷脂酶A1活性可水解两种磷脂类似物以释放理论量的游离硫醇，但磷脂酶C和A2均未促进硫的释放。测定了德氏根霉脂肪酶对每种底物水解的最适pH、热稳定性和Ca2+离子依赖性。结果表明，德氏根霉脂肪酶中的磷脂酶A1和单酰甘油脂肪酶活性是由不同的酶引起的，并且这些酶特异性测定对于酶的表征和纯化研究将具有普遍用途。这些底物可用于在pH范围3至8内对磷脂酶A1和单酰甘油脂肪酶进行灵敏、便捷且特异的分光光度法测定。
