PHOSPHOLIPASE C-PROMOTED MEMBRANE-FUSION - RETROINHIBITION BY THE END-PRODUCT DIACYLGLYCEROL

The catalytic activity of phospholipase C induces fusion of pure lipid vesicles. When large unilamellar liposomes composed of phosphatidylcholine/phosphatidylethanolamine/cholesterol (2:1:1 mole ratio) are treated with phospholipase C, in the presence of 10 mM Ca2+, two enzyme effects can be distinguished: a fast one (half-time on the order of seconds) consisting mainly of vesicle-vesicle fusion and a slow one (half-time on the order of minutes) representing bulk lipid hydrolysis. The fast fusion process is inhibited by the end-product diacylglycerol, as well as by lysophosphatidylcholine and by low Ca2+ concentrations. The temperature dependence of enzyme activity (phospholipid hydrolysis), vesicle aggregation, and vesicle fusion (mixing of aqueous contents) has been separately studied. Enzyme activity and vesicle aggregation rates increase monotonically with temperature, while an optimum temperature is found for vesicle fusion, depending on liposome composition and assay conditions. The presence of diacylglycerol incorporated to the membrane (up to 10 mol %) does not produce any fusion effect even at temperatures as high as 80-degrees-C: in situ diacylglycerol production by the enzyme appears to be required. The data are interpreted in support of a hypothesis according to which a ''fusion intermediate'' would be required, depending (among others) on bilayer composition, temperature, and Ca2+ concentration, for vesicle fusion to occur.