Dependence on neutral salt concentration of the latency phase in the time course of hydrolysis of dimyristoylphosphatidylcholine liposomes by phospholipase A₂

Abstract

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.

Enhancement of Agkistrodon piscivorus piscivorus venom phospholipase A2 activity toward phosphatidylcholine vesicles by lysolecithin and palmitic acid: Studies with fluorescent probes of membrane structure

“…Therefore, they could represent conditions in the membrane facilitating the increased PLA2 activity at . To improve the resolution of the experiment to verify this important result, we used high concentrations of KC1 to slow the hydrolysis time course (Fernández et al, 1991). Figure Figure 7 shows the effect of incremental addition of equimolar lyso-PC and PA to DPPC LUV on the emission spectrum of Prodan at 40 °C and 1 mM calcium (Figure 7A), 6 µ calcium (Figure 7B), or 1 µ calcium (Figure 7C).…”

Interfacial Enzymology: The Secreted Phospholipase A2-Paradigm

“…The concentration of the product on the enzyme-containing vesicles changes the position of the E to E* equilibrium during the reaction progress. Also, fusion of the product-containing vesicles is promoted by a millimolar concentration of calcium. , At some stage of this analysis it may be necessary to make assumptions about the effects of nonideal miscibility of the products 108,167 and the consequent changes in the phase properties of the bilayer during the reaction progress. − Also note that the delay to steady-state has also been accounted for by a kinetic model in which desorption of the enzyme from the interface is promoted by the products of hydrolysis,160b although the assumption is inconsistent with independent measurements. Considering the contributions of such variables along with the turnover parameters, the study of sPLA2s that display the long-delay phenomenon on zwitterionic phosphatidylcholine vesicles is unlikely to provide unequivocal information about the primary events.…”