Determination of the activation volume of PLCbeta by Gbeta gamma-subunits through the use of high hydrostatic pressure.
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.
Scarlata
Suzanne
S
Department of Physiology & Biophysics, State University of New York, Stony Brook, New York 11794-8661, USA. Suzanne.Scarlata@sunysb.edu
D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001703:Biophysics / Q000379:methods*; D002021:Buffers; D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry*; D007313:Insecta; D007527:Isoenzymes / Q000737:chemistry*; D008051:Lipid Bilayers; D008055:Lipids / Q000737:chemistry; D046911:Macromolecular Substances / Q000737:chemistry; D008566:Membranes / Q000737:chemistry; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D054799:Phospholipase C beta; D011312:Pressure; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D010738:Type C Phospholipases / Q000737:chemistry*
D002021:Buffers; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D008051:Lipid Bilayers; D008055:Lipids; D046911:Macromolecular Substances; D010455:Peptides; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011506:Proteins; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; D011493:Protein Kinase C; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine