Competitive inhibition of lipolytic enzymes. VIII: Inhibitor-induced aggregation of porcine pancreatic phospholipase A2.

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A2 (De Haas, G.H., Dijkman, R., Ransac, S. and Verger, R. (1990) Biochim. Biophys. Acta 1046, 249-257). Their inhibitory power appeared to be strictly controlled by the stereoconfiguration around the chiral C-2 atom and effective inhibition of the enzyme was observed only when incorporated into a micellar substrate-water interface. In the present study various direct binding techniques were applied to investigate the interaction of the enzyme with pure micelles of the stereoisomeric forms of 2-tetradecyl-amino-hexanol-1-phosphocholine (R-C14-PN and S-C14-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass = 14 kDa) on calibrated Superdex columns running in micellar solutions of R-C14-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa. Under identical conditions, micellar solutions of S-C14-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position. Light scattering experiments, ultrasedimentation and time-resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between enzyme and R-C14-PN micelles. The ultimate complex was shown to consist of four protein and about ten inhibitor molecules. Using time-resolved fluorescence spectroscopy the interaction was studied between the active site of phospholipase A2 and R-C14-PN molecules, both incorporated in an inert lipid matrix.