Preparation of Liposomes with Asymmetric Distribution of Phosphyatidylinositol 4,5-Bisphosphate Across the Bilayer

Abstract

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°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.