A model for the activation of porcine pancreatic phospholipase A₂ for the hydrolysis of dipalmitoylphosphatidylcholine liposomes

The hydrolysis of large unilamellar vesicles made of dipalmitoylphosphatidylcholine (DPPC) by porcine pancreatic phospholipase A₂ (PLA₂) have been studied as a function of temperature. The time courses of these reactions depend on the structural state of the phospholipid substrate. In neither the gel phase nor the liquid-crystalline phase, hydrolysis occurs, whereas in the phase transition region (39-42°C) the hydrolysis is characterized by a phase of slow hydrolysis (latency phase of a length of time τ), followed by an abrupt increase in the rate of hydrolysis. The latency (τ) is minimal at the phase transition temperature (T_m) in agreement with previous data, which suggested that activation requires the existence of packing irregularities. Such irregularities exist only in the transition region and maximize at the T_m. Preincubation of a mixture of enzyme and substrate vesicles at a temperature below T_m, when followed by dilution into a medium at the transition region, results in virtually instantaneous hydrolysis. This hydrolysis is independent of the presence of Ca²⁺ in the preincubation medium. These results are interpreted in terms of the following scheme for the hydrolysis of DPPC by PLA₂: First, the enzyme binds to the lipid bilayer in a Ca²⁺-independent step which occurs best for gel-phase lipids. This step is then followed by activation of the initially formed enzyme-substrate complex, which requires Ca²⁺ and can only occur if packing irregularities exist in the membrane (in the thermal transition range of the vesicles). Reaction products play a role in the hydrolysis at the T_m range by increasing the binding of PLA₂ to the vesicle surface. In this temperature range, increased binding is sufficient to cause virtually instantaneous hydrolysis, since activation is relatively fast.