Catecholamine-Stimulated Guanosine 5′-O-(3-Thiotriphosphate) Binding to the Stimulatory GTP-Binding Protein of Adenylate Cyclase: Kinetic Analysis in Reconstituted Phospholipid Vesicles

Tomiko Asano, Elliott M. Ross

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

The stimulatory GTP-binding protein of adenylate cyclase, Gs, and β-adrenergic receptors were reconstituted into unilamellar phospholipid vesicles. The kinetics of the quasi-irreversible binding of guanosine 5′-O-(3-thiotriphosphate) (Gtpγs) to Gs, equivalent to Gs activation by nucleotide, was studied with respect to the stimulation of this process by β-adrenergic agonists and Mg2+. The rate of Gtpγs binding displayed apparent first-order kinetics over a wide range of nucleotide, agonist, and Mg2+ concentrations. In the absence of agonist, the apparent first-order rate constant, kapp, was 0.17-0.34 min-1 and did not vary significantly with the concentration of nucleotide. At 50 mM MgCl2, kapp increased somewhat, to 0.26-0.41 min-1, and remained invariant with the nucleotide concentration. In the presence of agonist, kapp was dependent on nucleotide concentration. At 10-9 M Gtpγs, the addition of (-)-isoproterenol caused at most a 2-fold stimulation of kapp. However, kapp measured in the presence of isoproterenol increased as an apparently saturable function of the Gtpγs concentration, such that isoproterenol caused a 17-fold increase in kapp at 1 μM Gtpγs. The effect of isoproterenol on kapp also appeared to saturate at high isoproterenol concentration, yielding a kapp ~ 6 min-1 at high concentrations of both nucleotide and agonist. These data suggest that the receptor-agonist complex acts by increasing the rate of conversion of a lower affinity Gs-Gtpγs complex to the stable activated state.

Original languageEnglish (US)
Pages (from-to)5467-5471
Number of pages5
JournalBiochemistry
Volume23
Issue number23
DOIs
StatePublished - Nov 1984

ASJC Scopus subject areas

  • Biochemistry

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