Hydrolysis of phosphatidylinositol 4,5-bisphosphate in liposomal bilayers by phosphoinositide-specific phospholipase C.

Phosphatidylinositol 4,5-bisphosphate (PIP$\sb2$) constitutes only a minor fraction of the amount of phospholipids encountered in biomembranes. However, its role in the normal functioning of living cells is significant and has been shown to be increasing in importance. In order to gain a better understanding of the role and functions of PIP$\sb2$ in such processes and allow for methodologies that would facilitate control of such functions, it is necessary to generate an appropriate membrane model. Since PIP$\sb2$ is believed to be located preferentially on the inner leaflet of membrane bilayers, it is clear that a model membrane asymmetric with respect to the distribution of PIP$\sb2$ is essential. Phosphoinositide-specific phospholipase C (PI-PLC) is a promising tool for the generation of such asymmetric model membranes and also has potential value in quantitating PIP$\sb2$ distribution between the inner and outer leaflets of biological membranes. In this study, certain factors that are likely to influence the interactions of PI-PLC with PIP$\sb2$-containing liposomes were investigated. Symmetric PIP$\sb2$-containing liposomes were treated with enzyme extract activity partially purified from outdated human platelets and the PIP$\sb2$ hydrolysis profiles were monitored under a variety of experimental conditions. It was found that the size and composition of the substrate-containing liposomes, the specific activity and composition of the enzyme extracts, the pH of the incubation medium and the absolute amounts of the enzyme in the reaction mixture affect both the rate and extent or PIP$\sb2$ hydrolysis in the liposomal bilayers. Electrostatic effects mediated by the inclusion of a negatively charged lipid in the PIP$\sb2$-containing liposomal bilayer or by Ca$\sp{2+}$ ions in the incubation medium were of particular importance. Contaminant proteins in the enzyme preparations were shown to cause liposomal aggregation, thus reducing the extent of PIP$\sb2$ hydrolysis. These factors should be taken into consideration when PI-PLC is used to modify phospholipid compositions in bilayers of both artificial and biological membranes.