DIRECT IMAGING BY CRYO-TEM SHOWS MEMBRANE BREAK-UP BY PHOSPHOLIPASE A(2) ENZYMATIC-ACTIVITY

Phospholipid hydrolysis to free fatty acid and l-lyso-phospholipid by water-soluble phospholipase A(2) (PLA(2)) at the surface of lipid memb ranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kineti c phenomenon may increase our insight into the function of PLA2 under physiological conditions as well as into general interfacial catalysis . In the present study we apply for the first time cryo-transmission e lectron microscopy (cryo-TEM) and high-performance liquid chromatograp hy (HPLC) to characterize the PLA2 hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our dire ct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis, Most strikingly, c ryo-TEM reveals that starting in the lag phase, vesicles become perfor ated and degrade into open vesicles, bilayer fragments, and micelles, This structural instability extends throughout the system in the activ ity burst regime. In agreement with earlier reported correlations betw een initial phospholipase activity and substrate morphology, our resul ts suggest that the lag-burst phenomenon reflects a cascade process. T he PLA(2)-induced changes in lipid composition transform the morpholog y which in turn results in an acceleration of the rate of hydrolysis b ecause of a strong coupling between the PLA(2) activity and the morpho logy of the lipid suspension.