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

Polymerized liposomes of thiol-based phospholipids, (lipoyloxy)dodecan oyl]-sn-glycero-3-phosphocholine (BLPC) and -phosphoglycerol (BLPG) we re used to study interactions of several phospholipases A2 (PLA2) with membranes. Large liposomes (an average diameter of 100 +/- 10 nm) pre pared from BLPC or BLPG were readily hydrolyzed by PLA2. Once polymeri zed, however, these liposomes were resistant to the PLA2 hydrolysis. W hen liposomes were prepared from a mixture of -2-(1-pyrenyldecanoyl)-s n-glycero-3-phosphocholine (pyrene-PC) (5 mol %) and BLPC, fluorescenc e measurements of resulting polymerized mixed liposomes showed that th e pyrene-PC molecules exist solely as monomers without forming a patch and were selectively hydrolyzed by PLA2. Progress of the hydrolysis c an be readily monitored by measuring the change in fluorescence emissi on at 380 nm in the presence of bovine serum albumin. Rapid and select ive hydrolysis of inserted phospholipids in polymerized mixed liposome s supports the notion that facile migration of a phospholipid substrat e from membrane to the active site of enzyme is a critical step in the catalysis of PLA2. On the basis of these findings, various combinatio ns of polymerized mixed liposomes were prepared and their hydrolysis b y PLA2 measured. When compared to the substrate specificity of PLA2s d etermined 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 pla y an important role in the determination of substrate specificity of P LA2 and in the regulation of PLA2 activities. Lastly, polymerized mixe d liposomes can serve as a versatile and sensitive PLA2 assay system i n which one can readily modify the structure of polymerized matrix to create liposome surfaces ideal for a specific PLA2.