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

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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
StatePublished - 1984

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Guanosine 5'-O-(3-Thiotriphosphate)
GTP-Binding Proteins
Adenylyl Cyclases
Catecholamines
Phospholipids
Isoproterenol
Nucleotides
Kinetics
Unilamellar Liposomes
Adrenergic Agonists
Magnesium Chloride
Adrenergic Receptors
Rate constants
Chemical activation

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Catecholamine-stimulated guanosine 5′-O-(3-thiotriphosphate) binding to the stimulatory GTP-binding protein of adenylate cyclase: Kinetic analysis in reconstituted phospholipid vesicles",
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.",
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AU - Ross, Elliott M.

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