PHOSPHATIDYLCHOLINE ACTIVATION OF BACTERIAL PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE-C TOWARD PI VESICLES

The effect of different phospholipids on the kinetic behavior of phosp hatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thurin giensis toward PI vesicles has been investigated. Cosonicated PC/PI ve sicles displayed enhanced hydrolysis of PI when less than 0.20 mole fr action PC was incorporated into the vesicle; higher mole fractions of PC led to a decrease from the maximum activity mimicking surface dilut ion of substrate. Since the PC could affect PI-PLC binding to vesicles , the effect of separate PC vesicles on enzymatic hydrolysis of PI ves icles was examined. Separate phosphatidylcholine vesicles were found t o activate PI-PLC-catalyzed cleavage of PI vesicles up to 7-fold. The activation was completely abolished when the PC vesicle was composed o f cross-linked molecules. In the absence of enzyme, fluorescence reson ance energy transfer studies did not detect any fusion between PI and PC vesicles if the total lipid concentration was below 2 mM. Higher to tal lipid concentrations (>20 mM> increased PC transfer between PC and PI vesicles, producing a PI vesicle population with small amounts of PC in the outer monolayer. This suggested that the activation of PI-PL C toward PI vesicles reflects the time scale of transfer of PC from PC vesicles to PI vesicles. Cosonicated PC/PI vesicles provide a measure of enzyme activity versus mole fraction of PC that can be used to est imate the extent of vesicle exchange or fusion between separate vesicl e pools. The effects of other phospholipid vesicles on PI-PLC hydrolys is of PI were also examined; zwitterionic lipids were activators while anionic phospholipids inhibited activity. The results indicated that PC molecules in the PI interface allosterically bind to PI-PLC and hel p anchor enzyme in a more active conformation to the PI interface.