Determination of the Catalytic Activity of Phospholipase A2: E. coli-Based Assay Compared to a Photometric Micelle Assay

Abstract

Summary: Phospholipase A 2 activity in human sera was determined of the basis of the E. coli assay and compared to a photometric micelle assay. The E. coli assay is based on the hydrolysis of phospholipids from [l-14 C]oleic acid-labelled E. coli biomembranes. In the photometric assay the phospholipase A 2 acts on mixed phospholipid micelles. The amount of fatty acid produced is quantitated in a subsequent photometric assay by coupling in the reaction to the coenzyme A metabolism. The E. coli membranes are essentially resistent to other Upases in human sera, i. e. lipoprotein upases, hepatic triacylglycerolipase or pancreatic lipase and thus a very specific substrate for the phospholipase A 2 of human serum. The photometric assay, though, is susceptible to other upases in human serum. The ratio of (l-14 C]oleic acid to released total fatty acids served as the basis for the calculation of the true enzymatic activity. The assay closely correlated with the photometric assay based on mixed micelles in the higher ranges of phospholipase A 2 activity, but not in the normal range. The sensitivity is higher by at least two powers of 10. The human serum phospholipase A 2 strongly preferred E. coli membranes as substrate to the mixed micelles containing phosphatidylcholine/phosphatidylethanolamine. In conclusion, the modified phospholipase A 2 assay based on E. coli membranes is a sensitive, specific, reliable, and convenient method for the measurement of phospholipase A 2 activity in human sera. The photometric assay süffers from low sensitivity but has the advantage of practicability in a normal routine laboratory, including the amenability to automation. Introduction... . , , , ,

Phospholipase A2 activity of crocodile serum

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