PHOSPHOLIPID ACTIVATION OF COBRA VENOM PHOSPHOLIPASE-A2 .1. LIPID-LIPID OR LIPID-ENZYME INTERACTION

In individual phospholipid-Triton X-100 mixed micelles, phospholipase A2 hydrolyzes phosphatidylcholine at a tenfold higher rate than phosphatidylethanolamine, while in binary phospholipid mixtures phosphatidylcholine activates the enzyme toward phosphatidylethanolamine so that it becomes the preferred substrate. This specificity reversal has now been observed in anionic and zwitterionic as well as in nonionic detergent mixed micelle systems. Detailed studies on the physical characteristics of Triton X-100/phospholipid mixed micelles were conducted to ascertain their role in the activation process. The size of Triton mixed micelles containing phosphatidylcholine, phosphatidylethanolamine, or cholesterol, individually and in combination, was determined by agarose gel chromatography. Micelle size depends only on the total mole fraction of lipid, not on the phospholipid head group or whether the lipid is cholesterol, and the Stokes radius increases by about one-third in going from a mole fraction of lipid of 0.06 to 0.20. 31P nuclear magnetic resonance spectroscopy was used to study lipid hydrolysis by phospholipase A2 and to determine the importance of lipid-lipid interactions. Preferential hydrolysis of phosphatidylethanolamine is independent of the mole ratio of Triton/phospholipid and the presence of cholesterol. The 31P{1H) nuclear Overhauser effect enhancements for phosphatidylcholine and phosphatidylethanolamine alone and for the two in binary lipid mixed micelles have the same frequency dependence, maximum at methylene protons adjacent to the phosphate, suggesting that no strong intermolecular lipid head-group interactions occur. These data demonstrate that mixed micelle size, shape, surface charge, and lipid-lipid interactions are not responsible for specificity reversal. The observation that the soluble, monomeric phospholipid dibutyrylphosphatidylcholine, which is shown to not form mixed micelles with Triton, activates phospholipase A2 toward phosphatidylethanolamine suggests a direct lipid-enzyme interaction as the cause of the activation process. © 1979, American Chemical Society. All rights reserved.