Kinetic analysis of phospholipase C from catharanthus roseus transformed roots using different assays

The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [(33)P]phosphatidylinositol 4,5-bisphosphate (PIP(2)) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP(2) hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP(2) vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP(2) and PIP(2) surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 &mgr;M. These findings will add to our understanding of the mechanisms of regulation of plant PLC.