Development of an assay for phospholipase C using column-reconstituted, extruded phospholipid vesicles.

The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-beta activity in vitro. We have developed an improved and sensitive method for the assay of PLC-beta a ctivity. This approach involves reconstitution of purified beta gamma dimer s into extruded phospholipid vesicles containing phosphatidylinositol 4,5-b isphosphate and using a gel-filtration technique to separate the reconstitu ted vesicles from monodispersed beta gamma dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of beta gamma dimers into phospholipid vesicles and was used t o examine the effect of different prenyl groups on the gamma subunits in th e activation of PLC-beta. The beta(1)gamma(1) dimer (containing the farnesy l group) and the beta(1)gamma(2) dimer (containing the geranylgeranyl group ) were purified from baculovirus-infected Sf9 insect cells and were found t o partition equally into phospholipid vesicles. The beta(1)gamma(2) dimer i s more potent and effective in stimulating PLC-beta activity than the beta( 1)gamma(1) dimer. The EC50 values of beta gamma dimers for the activation o f PLC-beta determined with this method were lower than those determined by previous methodology, showing that beta gamma subunits have a subnanomolar affinity for PLC-beta. (C) 1999 Academic Press.