USE OF POLYMERIZED MIXED LIPOSOMES TO STUDY INTERACTIONS OF PHOSPHOLIPASE-A(2) WITH MEMBRANES

Polymerized liposomes of thiol-based phospholipids, 1,2-bis[12-(lipoyloxy)dodecanoyl]-sn-glycero-3-phosphocholine (BLPC) and -phosphoglycerol (BLPG) were used to study interactions of several phospholipases A2 (PLA2) with membranes. Large liposomes (an average diameter of 100 +/- 10 nm) prepared from BLPC or BLPG were readily hydrolyzed by PLA2. Once polymerized, however, these liposomes were resistant to the PLA2 hydrolysis. When liposomes were prepared from a mixture of 1-hexadecanoyl-2-(1-pyrenyldecanoyl)-sn-glycero-3-phosphocholine (pyrene-PC) (5 mol %) and BLPC, fluorescence measurements of resulting polymerized mixed liposomes showed that the pyrene-PC molecules exist solely as monomers without forming a patch and were selectively hydrolyzed by PLA2. Progress of the hydrolysis can be readily monitored by measuring the change in fluorescence emission at 380 nm in the presence of bovine serum albumin. Rapid and selective hydrolysis of inserted phospholipids in polymerized mixed liposomes supports the notion that facile migration of a phospholipid substrate from membrane to the active site of enzyme is a critical step in the catalysis of PLA2. On the basis of these findings, various combinations of polymerized mixed liposomes were prepared and their hydrolysis by PLA2 measured. When compared to the substrate specificity of PLA2s determined using Triton X-100/phospholipid mixed micelles, results from polymerized mixed liposomes indicate that electrostatic interactions between the interfacial binding site of PLA2 and membrane surfaces play an important role in the determination of substrate specificity of PLA2 and in the regulation of PLA2 activities. Lastly, polymerized mixed liposomes can serve as a versatile and sensitive PLA2 assay system in which one can readily modify the structure of polymerized matrix to create liposome surfaces ideal for a specific PLA2.