Preparation of liposomes with asymmetric distribution of phosphatidylinositol 4,5-bisphosphate across the bilayer

Asymmetric liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) predominantly on the inner leaflet of the bilayer were prepared by means of a non-penetrating enzyme, phosphoinositide-specific phospholipase C isolated from adult human platelets. Symmetric liposomes prepared by a modification of the reverse-phase evaporation method were incubated with partially purified enzyme in the presence of Ca2+ at 37 degrees C. The resultant liposomes were collected by high-speed centrifugation. Hydrolysis of PIP2 on the outer leaflet of the membrane was completed after approximately 4 h of incubation. Since PIP2 is predominantly located in the inner leaflet of biological membranes, these asymmetric liposomes should be suitable for investigation of structural and functional roles of PIP2 in biomembranes.