Kinetic control of guanine nucleotide binding to soluble Gα(q)

Vladislav S. Markin, Elliott M. Ross

Research output: Contribution to journalArticlepeer-review

43 Scopus citations

Abstract

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 Gα(q) displays unusual guanine nucleotide binding properties in comparison with other G protein α subunits. Under conditions where most G proteins bind nearly stoichiometric GTPγS in 5-30 min at micromolar nucleotide concentrations, GTPγS binding to Gα(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, GTPγS dissociation is immeasurably slow under commonly used conditions. We found that purified Gα(q) can bind stoichiometric GTPγS, but that binding is controlled kinetically by a combination of factors. GDP (or IDP) dissociated slowly from Gα(q), but the dissociation rate increased linearly with the concentration of (NH4)2SO4 up to 0.75 M (~20-fold acceleration). The resulting GDP-free Gα(q) was labile to rapid and irreversible denaturation, however (rate constant ≥ 1 min-1 at 20°). Denaturation competed kinetically with relatively slow GTPγS association, such that stoichiometric binding was only attained at 100 μM GTPγS. These findings reconcile the slowly reversible binding of GTPγS to Gα(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 α subunits. Understanding these interactions allowed the direct, accurate quantitation of active Gα(q) by a simple GTPγS binding assay in the presence of (NH4)2SO4, and similarly can prevent underestimation of the concentrations of other G proteins. Copyright (C) 1999 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)39-48
Number of pages10
JournalBiochemical Pharmacology
Volume58
Issue number1
DOIs
StatePublished - Jul 1 1999

Keywords

  • G protein
  • GTP-binding protein
  • Gα(q)
  • guanosine 5'-O-thiotriphosphate

ASJC Scopus subject areas

  • Biochemistry
  • Pharmacology

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