Hydrolysis of 1,2-diglyceride by membrane-associated lipase activity during phospholipase C treatment of membranes

1. 1. When rat erythrocyte membranes were treated with high concentrations of phospholipase C, approximately 70% of the phospholipids were hydrolysed. 2. 2. The main product of phospholipase C action, 1,2-diglyceride, did not accumulate even when 50% of the membrane phospholipid was hydrolysed in about 10 min. 3. 3. During phospholipase treatment unesterified fatty acids and a variable amount of monoglyceride accumulated. Between 1.2 and 1.8 ester bonds were cleaved per molecule of phospholipid hydrolysed by the phospholipase C, suggesting that the membranes contained a highly active lipase capable of attacking 1,2-diglyceride and monoglyceride. 4. 4. Hydrolysis of emulsified rat erythrocyte lipids with phospholipase C yielded 1,2-diglyceride but there was no hydrolysis of acyl ester bonds or liberation of unesterified fatty acids. Thus the lipase was not a contaminant in the phospholipase C preparations. 5. 5. Rat erythrocyte membranes treated with phospholipase C dereased in size, developed lipid droplets on their surface and remained morphologically intact. 6. 6. Hydrolysis of diglyceride generated in membrnes by phospholipase C action was observed (in order of decreasing activity) in rat erythrocyte membranes, in microsomes, plasma membranes and other subcellular fractions from rat liver and in pig erythrocyte membranes, human erythrocyte membranes, rat muscle microsomes and myelin. In the latter three preparations only a small fraction of the diglyceride was hydrolysed. 7. 7. These results are discussed in relation to the extensive use of phospholipase C in studies of the structure of biological membranes.