Analysis of the time course of hydrolysis of dimyristoylphosphatidylcholine liposomes catalyzed by porcine pancreatic phospholipase A2 at 18 degrees C shows that, in the presence of 10 mM NaCl, the length of the latency period in the presteady-state phase increases from 3 to 10.5 min when the CaCl2 concentration is reduced from 15 to 1 mM. This inverse dependence of the lag period on calcium ion concentration is seen more readily at 1 M NaCl, where the induction time changes from 13.5 to 42 min by decreasing the concentration of CaCl2 from 15 to 1 mM. To interpret these results, we took into account the small amount of fatty acid that is produced during the latency phases. The fatty acid generates a negative surface electrostatic potential and makes the interfacial concentration of calcium ions different from the concentration in the bulk solvent. Variations in the analytical concentrations of NaCl and CaCl2 affect both the interfacial calcium ion concentration and electrostatic potential, as estimated theoretically from Grahame and Boltzmann equations. According to these estimates, the length of the latency period diminishes with the increase of the interfacial calcium concentration, but does not show any logical dependence on the change in surface electrostatic potential.
The time course of the hydrolytic action of porcine pancreatic phospholipase A2 on sonicated dimyristoylphosphatidylcholine liposomes in the presence of variable NaCl concentrations has been studied at temperatures between 17 and 36 degrees C; at these temperatures liposomes are in the gel phase. At a NaCl concentration of 10 mM, the hydrolysis shows a small and constant lag period of 6-8 min at all temperatures within this range. As the temperature is raised into the liquid crystalline range, the latency phase lengthens monotonically so that at 36 degrees C it reaches 55 min. An increase in the NaCl concentration to 1 M makes the lag period longer at all temperatures studied, with the exception of the phase transition range (near 24 degrees C); within this temperature range, a small reduction in the lag time is observed. The increase in the length of the latency period at high salt concentrations may be due to screening of the negative surface charge generated by the nascent fatty acid which seems to be essential for the efficient interfacial binding of the enzyme. In the phase transition range of the lamellae, the unfavorable effect of high salt concentrations on the electrostatic binding of the enzyme appears to be overcome by another type of interaction. Recent findings raise the possibility that this interaction could be hydrophobic in nature.
SUMMARYPorcine pancreatic phospholipase Az (PLA2) has been incubated for 2 hours at 34~ with rabbit anti-pig pancreatic PLA2 polyclonal antiserum in the absence or presence of phosphatidylcholine liposomes in different physical states. Subsequent assay of hydrolysis -triggered through addition of 5 mM Ca 2+-at 34~ shows that preincubation with antiserum in the presence of 1,2-dimyristoyl-3-snphosphatidylcholine liquid-crystalline vesicles, renders the PLA2 activity undetectable, similarly to what is found if preincubation is carried out in the absence of liposomes. In contrast, 1,2-dipalmitoyl-3-snphosphatidylcholine liposomes, which at 34~ are in the gel-phase, protect the enzyme from the antiserum effect. The results are consistent with a stronger binding of PLA2 to gel phase, as compared to liquid crystalline vesicles and suggest that through the physical interaction with liposomes in the gel state, the enzyme is shielded from reaction with the antibodies. Taking into account the characteristic hydrolysis profiles of vesicles in different physical states, it can be concluded that the above interpretation agrees with the proposal that PLA2 -membrane association promotes the interracial activation of the enzyme.
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