The temporal sequence of events in the activation of phospholipase A2 by lipid vesicles. Studies with the monomeric enzyme from Agkistrodon piscivorus piscivorus.

The substrate dependence of the time courses of hydrolysis of both small and large unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) by Agkistrodon piscivorus piscivorus monomeric phospholipase A2 is consistent with an activation process involving enzyme aggregation on the vesicle surface. The time course of hydrolysis of large unilamellar vesicles is particularly complex; a slow initial rate of hydrolysis is followed by an extremely abrupt increase in enzyme activity. The length of this slow phase is a minimum at the phase transition temperature of the vesicles. The intrinsic fluorescence intensity of the phospholipase A2 also abruptly increases (50-60%) after a latency period revealing a strong temporal correlation between enzyme activity and the increase in fluorescence intensity. The length of the latency period before the sudden increase in fluorescence intensity is directly proportional to substrate concentration at DPPC concentrations above 20-100 microM. At lower concentrations, the length of the latency period is inversely proportional to the DPPC concentration. Such biphasic substrate dependence is predicted by a previously proposed enzyme activation model involving dimerization on the surface vesicle. Simultaneous monitoring of the protein fluorescence and hydrolysis demonstrates that the magnitude of the fluorescence change and the rate of hydrolysis are in exact temporal correlation. Furthermore, simultaneous monitoring of the fluorescence of the protein and that of a lipid probe, trimethylammonium diphenylhexatriene, indicates a change in lipid vesicle structure prior to, or coincident with, the abrupt change in protein activation. These results are consistent with the hypothesis that the monomeric phospholipase A2 from A. piscivorus piscivorus initially possesses a low level of intrinsic activity toward large unilamellar DPPC vesicles and that the enzyme slowly becomes further activated on the vesicle surface via dimerization. Eventually, the vesicles undergo an abrupt transition in internal structure leading to sudden rapid activation of the enzyme.