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Biophys J. 2005 Apr;88(4):2867-74. Epub 2005 Jan 21.

Determination of the activation volume of PLCbeta by Gbeta gamma-subunits through the use of high hydrostatic pressure.

Author information

1
Department of Physiology & Biophysics, State University of New York, Stony Brook, New York 11794-8661, USA. Suzanne.Scarlata@sunysb.edu

Abstract

Activation of phospholipase Cbeta (PLCbeta) by G-proteins results in increased intracellular Ca(2+) and activation of protein kinase C. We have previously found that activated PLCbeta-Gbetagamma complex can be rapidly deactivated by Galpha(GDP) subunits without dissociation, which led to the suggestion that Galpha(GDP) binds to PLCbeta-Gbeta gamma and perturbs the activating interaction without significantly affecting the PLCbeta-Gbeta gamma binding energy. Here, we have used high pressure fluorescence spectroscopy to determine the volume change associated with this interaction. Since PLCbeta 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: PLCbeta binding was stable throughout the 1-2000 bars range, Gbeta gamma binding was stable only at high membrane concentrations, whereas Galpha(s)(GDP) dissociated from membranes above 1 kbar. High pressure dissociated PLCbeta-Gbeta gamma with a DeltaV = 34 +/- 5 ml/mol. This same volume change is obtained for a peptide derived from Gbeta which also activates PLCbeta. In the presence of Galpha(s)(GDP), the volume change associated with PLCbeta-Gbeta gamma interaction is reduced to 25 +/- 1 ml/mol. These results suggest that activation of PLCbeta by Gbeta gamma is conferred by a small (i.e., 3-15 ml/mol) volume element.

PMID:
15665133
PMCID:
PMC1305381
DOI:
10.1529/biophysj.104.055715
[Indexed for MEDLINE]
Free PMC Article

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