Determination of the Activation Volume of PLCβ by Gβγ-Subunits through the Use of High Hydrostatic Pressure

Activation of phospholipase Cβ (PLCβ) by G-proteins results in increased intracellular Ca2+ and activation of protein kinase C. We have previously found that activated PLCβ-Gβγ complex can be rapidly deactivated by Gα(GDP) subunits without dissociation, which led to the suggestion that Gα(GDP) binds to PLCβ-Gβγ and perturbs the activating interaction without significantly affecting the PLCβ-Gβγ binding energy. Here, we have used high pressure fluorescence spectroscopy to determine the volume change associated with this interaction. Since PLCβ 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: PLCβ binding was stable throughout the 1–2000 bars range, Gβγ binding was stable only at high membrane concentrations, whereas Gαs(GDP) dissociated from membranes above 1 kbar. High pressure dissociated PLCβ-Gβγ with a ΔV = 34 ± 5 ml/mol. This same volume change is obtained for a peptide derived from Gβ which also activates PLCβ. In the presence of Gαs(GDP), the volume change associated with PLCβ-Gβγ interaction is reduced to 25 ± 1 ml/mol. These results suggest that activation of PLCβ by Gβγ is conferred by a small (i.e., 3–15 ml/mol) volume element.