1- 14C]oleate-labeled autoclaved yeast: A membranous substrate for measuring phospholipase A 2 activity in vitro

Abstract

Radiolabeled, autoclaved yeast were tested as a substrate for mammalian phospholipase A 2 activity because the only other membranous substrate used for this purpose, autoclaved Escherichia coli, totally lacks a major mammalian phospholipid, phosphatidylcholine. Candida albicans were grown in the presence of [1- 14C]oleate and then autoclaved. Sixty three percent of the incorporated label was in yeast phospholipid, and more than 95% of that was in the 2-acyl position. The distribution of label in the yeast phospholipids (phosphatidylcholine and -ethanolamine, -serine + -inositol, and phosphatidic acid corresponded closely to the chemical distribution of phosphorus in those phospholipids. Snake venom ( Naja naja) and human synovial fluid phospholipase A 2 hydrolyzed yeast phospholipid exclusively to release 14C-labeled fatty acid. When 50–60% of the yeast phospholipid was hydrolyzed, the radioactive fatty acids as determined by gas-liquid chromatographic analysis were predominantly oleate (45%) and linoleate (>54%). Hydrolysis of yeast phospholipid by both enzymes was near-linear with protein and time under conditions of optimal pH (neutral-alkaline) and Ca 2+ (1–5 m m) previously reported for optimal hydrolysis of autoclaved E. coli phospholipid. N. naja phospholipase A 2 showed less preference for phosphatidyleth-anolamine than -choline as liposomes or yeast phospholipid as compared to human synovial fluid phospholipase A 2 which clearly preferred phosphatidylethanolamine to -choline as a liposome or yeast phospholipid. These results illustrate that radiolabeled phospholipids of autoclaved yeast, enriched in phosphatidylcholine, are readily hydrolyzed by snake venom and human nonpancreatic phospholipases A 2 and may, therefore, be useful in the measurement of in vitro enzymatic activity.