Figure 1.

Schematic of PI-PLC assay. Incubation of unilamellar liposomes composed of egg PC and egg PA (9:1, molar ratio; green symbols) and trace amounts of [³H]PI (red symbols) with PI-PLC results in hydrolysis of [³H]PI (to inositol cyclic phosphate and diacylglycerol (DAG)) in the outer leaflet of the bilayer, whereas [³H]PI in the inner leaflet is protected from the enzyme. Because [³H]PI distributes equally between the two leaflets during preparation of liposomes, ∼50% is expected to be hydrolyzed by PI-PLC. In unilamellar proteoliposomes reconstituted with scramblase protein(s), [³H]PI from the inner leaflet is translocated to the outer leaflet, where it becomes accessible to PI-PLC. The extent of [³H]PI hydrolysis in scramblase-containing proteoliposomes is expected to reach 100% on a relatively short time scale.