Determination of the activation volume of PLCß by Gßγ-subunits through the use of high hydrostatic pressure

Activation of phospholipase C beta (PLC beta) by G-proteins results in increased intracellular Ca2+ and activation of protein kinase C. We have previously found that activated PLC beta-G beta gamma complex can be rapidly deactivated by G alpha(GDP) subunits without dissociation, which led to the suggestion that G alpha(GDP) binds to PLC beta-G beta gamma and perturbs the activating interaction without significantly affecting the PLC beta-G beta gamma binding energy. Here, we have used high pressure fluorescence spectroscopy to determine the volume change associated with this interaction. Since PLC beta and G-protein subunits associate on membrane surfaces, we worked under conditions where the membrane surface properties are not expected to change. We also determined the pressure range in which the proteins remain membrane bound: PLC beta binding was stable throughout the 1-2000 bars range, G beta gamma binding was stable only at high membrane concentrations, whereas G alpha(s)(GDP) dissociated from membranes above 1 kbar. High pressure dissociated PLC beta-G beta gamma with a Delta V = 34 +/- 5 ml/mol. This same volume change is obtained for a peptide derived from G beta which also activates PLC beta. In the presence of G alpha(s)(GDP), the volume change associated with PLC beta-G beta gamma interaction is reduced to 25 +/- 1 ml/mol. These results suggest that activation of PLC beta by G beta gamma is conferred by a small (i.e., 3-15 ml/mol) volume element.