Kinetic control of guanine nucleotide binding to soluble Galpha(q).
Binding of guanine nucleotides to heterotrimeric G proteins is controlled primarily by kinetic factors, such as the release of bound GDP, rather than by affinity alone. Detergent-solubilized Galpha(q) displays unusual guanine nucleotide binding properties in comparison with other G protein alpha subunits. Under conditions where most G proteins bind nearly stoichiometric GTPgammaS in 5-30 min at micromolar nucleotide concentrations, GTPgammaS binding to Galpha(q) is slow (>1 hr to completion), markedly substoichiometric, and dependent upon high concentrations of nucleotide (0.1 to 0.2 mM). Although the latter two properties suggest low affinity, GTPgammaS dissociation is immeasurably slow under commonly used conditions. We found that purified Galpha(q) can bind stoichiometric GTPgammaS, but that binding is controlled kinetically by a combination of factors. GDP (or IDP) dissociated slowly from Galpha(q), but the dissociation rate increased linearly with the concentration of (NH4)2SO4 up to 0.75 M (approximately 20-fold acceleration). The resulting GDP-free Galpha(q) was labile to rapid and irreversible denaturation, however (rate constant > or = 1 min(-1) at 20 degrees). Denaturation competed kinetically with relatively slow GTPgammaS association, such that stoichiometric binding was only attained at 100 microM GTPgammaS. These findings reconcile the slowly reversible binding of GTPgammaS to Galpha(q) with the other behaviors that suggested lower affinity, and point out that events subsequent to GDP dissociation can markedly influence the rates and extents of guanine nucleotide binding to G protein alpha subunits. Understanding these interactions allowed the direct, accurate quantitation of active Galpha(q) by a simple GTPgammaS binding assay in the presence of (NH4)2SO4, and similarly can prevent underestimation of the concentrations of other G proteins.
Chidiac
P
P
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.
Markin
V S
VS
Ross
E M
EM
D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D007313:Insecta; D007700:Kinetics; D008024:Ligands; D051379:Mice; D011485:Protein Binding; D011489:Protein Denaturation; D013462:Sulfur Radioisotopes; D013997:Time Factors
D006150:Guanine Nucleotides; D008024:Ligands; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D000645:Ammonium Sulfate