Pulmonary surfactant isolated by lavage can be separated into large aggregates (LA) and small aggregates (SA). Pulse labeling experiments have shown that the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitr

Group IIA and V phospholipases A2 (PLA2s) are known to play a role in inflammatory responses. We have constructed a bacterial expression vector for rat group IIA and V PLA2s, over-expressed, folded and purified the proteins with the aim to study and compare the properties of the enzymes in detail. For zwitterionic phospholipid micelles, both enzymes display optimum activity at pH 8.0 and absolutely require Ca2+ for enzymatic activity. In the presence of substrate, group V PLA2 has a high affinity for Ca2+ (KCa2+=90 μM) while KCa2+ of group IIA PLA2 was found to be 1.6 mM. The absence of substrate only marginally influences the Ca2+ affinities. In contrast to group IIA PLA2, group V PLA2 does not show a jump in the activity profile at substrate concentrations around the critical micelle concentration. Direct binding studies using n-alkylphosphocholines indicate that group V PLA2 forms protein-lipid aggregates at pre-micellar lipid concentrations in a cooperative and Ca2+-dependent manner. This behavior, which is comparable to that observed for the PLA2 from Naja melanoleuca snake venom, reflects the high affinity of this enzyme for zwitterionic phospholipids. Competitive inhibition by the substrate analogues (R)-2-dodecanoylaminohexanol-1-phosphocholine and its phosphoglycol derivative was tested on zwitterionic micelles as substrate. Group IIA PLA2 shows a preference for the phosphoglycol inhibitor whereas the phosphocholine inhibitor binds stronger to the active site of group V PLA2. The enzymatic activity was also measured on zwitterionic liposomes which appear to be much better substrates for group V PLA2 than for group IIA PLA2. The overall results suggest that group V PLA2 is better suited for action on biological membranes than group IIA PLA2.