Reversibility of the activation of soluble phospholipase A 2 on lipid bilayers: Implications for the activation mechanism

Abstract

The time-courses of hydrolysis of large vesicles of dipalmitoylphosphatidylcholine were compared using four species of phospholipase A 2 ( Agkistrodon piscivorus piscivorus, Crotalus adamanteus and Naja naja venoms and porcine pancreatic). In all four cases, the hydrolysis rate suddenly increases 10 to 100-fold at the time (τ) when a specific mole fraction of reaction products has accumulated. The intrinsic fluorescence emission of the three venom enzymes also increases suddenly at time τ. Both the activation and the fluorescence change are reversible with a half-time of about 50 s for the activity and 2 to 6 s for the fluorescence. These reversal rates and the vesicle concentration dependence of τ are considered for monomer and dimer enzyme activation models. Apparently, at least three states of the enzyme exist beyond the initial unbound state: (1) inactive and bound, (2) inactive with high fluorescence and (3) active. The dimer model already contains the necessary number of states but requires that the activation rate be much lower than the reversal rate to account for the vesicle concentration dependence of τ. Success of the monomer model requires an enzyme state additional to those proposed previously. Although these results do not exclude either the monomer or dimer models conclusively, they do impose important constraints on each model.