PHOSPHOLIPASE-C BETA-2 ASSOCIATION WITH PHOSPHOLIPID INTERFACES ASSESSED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER - G-PROTEIN BETA-GAMMA SUBUNIT-MEDIATED TRANSLOCATION IS NOT REQUIRED FOR ENZYME ACTIVATION

Phospholipase C beta 2 (PLC beta 2) is activated by G protein beta gam ma subunits and calcium. The enzyme is soluble and its substrate, phos phatidylinositol 4,5-bisphosphate (PIP2), is present in phospholipid m embranes. A potential mechanism for regulation of this enzyme is throu gh influencing the equilibrium association of the enzyme with membrane surfaces. In this paper we describe a fluorescence resonance energy t ransfer (FRET) method for measuring the association of PLC beta 2 with phospholipid bilayers. The method allows equilibrium measurements to be made under a variety of conditions, including those that support en zymatic activity and ability to be regulated by G proteins. Using this method it was found that PLC beta 2 bound to vesicles containing anio nic lipids and demonstrated a selective and unique interaction with PI P2 containing vesicles. The FRET data were corroborated with a centrif ugation based method for estimating the affinity of PLC beta 2 for ves icles. Apparently different modes of association of PLC beta 2 with ve sicles of different composition can be distinguished based on alterati ons in resonance energy transfer efficiency. Association of PLC beta 2 with PLP(2) vesicles requires an intact lipid bilayer, is blocked by neomycin, and is not affected by D-myo-inositol 1,4,5-trisphosphate (D -IP3). G protein beta gamma subunits do not alter the affinity of PLC beta 2 for lipid bilayers and at the PIP2 concentrations used to measu re beta gamma-dependent stimulation of PLC activity, the majority of t he PLC beta 2 is already associated with the vesicle surface. Furtherm ore, under conditions where beta gamma subunits strongly activate PLC activity, the extent of association with vesicles is unaffected by bet a gamma subunits or calcium. These results indicate that activation of PLC beta 2 by G protein py subunits or Ca2+ in vitro does not involve translocation to the vesicle surface.