DEVELOPMENT OF AN ASSAY FOR PHOSPHOLIPASE C USING COLUMN-RECONSTITUTED, EXTRUDED PHOSPHOLIPID VESICLES

Abstract The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-β activity in vitro. We have developed an improved and sensitive method for the assay of PLC-β activity. This approach involves reconstitution of purified βγ dimers into extruded phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate and using a gel-filtration technique to separate the reconstituted vesicles from monodispersed βγ dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of βγ dimers into phospholipid vesicles and was used to examine the effect of different prenyl groups on the γ subunits in the activation of PLC-β. The β 1 γ 1 dimer (containing the farnesyl group) and the β 1 γ 2 dimer (containing the geranylgeranyl group) were purified from baculovirus-infected Sf9 insect cells and were found to partition equally into phospholipid vesicles. The β 1 γ 2 dimer is more potent and effective in stimulating PLC-β activity than the β 1 γ 1 dimer. The EC 50 values of βγ dimers for the activation of PLC-β determined with this method were lower than those determined by previous methodology, showing that βγ subunits have a subnanomolar affinity for PLC-β.