Rapid binding of guanosine 5′-O-(3-thiotriphosphate) to an apparent complex of β-adrenergic receptor and the GTP-binding regulatory protein Gs

David C. May, Elliott M. Ross

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17 Citations (Scopus)

Abstract

When reconstituted phospholipid vesicles that contain purified β-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), the typical receptor-stimulated GTPγS binding reaction was preceded by an even more rapid burst of GTPγS binding. This burst was studied in detail at 0°C. The rate of the burst was second order in nucleotide and Gs [kassoc∼ 2 × 107 (M·min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 ∼15 min at 0°C in the presence of agonist and decayed by ∼3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at 30°C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.

Original languageEnglish (US)
Pages (from-to)4888-4893
Number of pages6
JournalBiochemistry
Volume27
Issue number13
StatePublished - 1988

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Guanosine 5'-O-(3-Thiotriphosphate)
Guanine Nucleotides
Guanosine Triphosphate
GTP-Binding Proteins
Detergents
Adrenergic Receptors
Phospholipids
Nucleotides
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Rapid binding of guanosine 5′-O-(3-thiotriphosphate) to an apparent complex of β-adrenergic receptor and the GTP-binding regulatory protein Gs",
abstract = "When reconstituted phospholipid vesicles that contain purified β-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), the typical receptor-stimulated GTPγS binding reaction was preceded by an even more rapid burst of GTPγS binding. This burst was studied in detail at 0°C. The rate of the burst was second order in nucleotide and Gs [kassoc∼ 2 × 107 (M·min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 ∼15 min at 0°C in the presence of agonist and decayed by ∼3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at 30°C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.",
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T1 - Rapid binding of guanosine 5′-O-(3-thiotriphosphate) to an apparent complex of β-adrenergic receptor and the GTP-binding regulatory protein Gs

AU - May, David C.

AU - Ross, Elliott M.

PY - 1988

Y1 - 1988

N2 - When reconstituted phospholipid vesicles that contain purified β-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), the typical receptor-stimulated GTPγS binding reaction was preceded by an even more rapid burst of GTPγS binding. This burst was studied in detail at 0°C. The rate of the burst was second order in nucleotide and Gs [kassoc∼ 2 × 107 (M·min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 ∼15 min at 0°C in the presence of agonist and decayed by ∼3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at 30°C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.

AB - When reconstituted phospholipid vesicles that contain purified β-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), the typical receptor-stimulated GTPγS binding reaction was preceded by an even more rapid burst of GTPγS binding. This burst was studied in detail at 0°C. The rate of the burst was second order in nucleotide and Gs [kassoc∼ 2 × 107 (M·min)-1], consistent with diffusion-controlled binding. The magnitude of the burst was always less than the number of receptors present and was roughly linear with receptor number when similarly prepared vesicles were compared. There was no obvious quantitative correlation between the burst and the amount of Gs. The species that gave rise to the burst formed with t1/2 ∼15 min at 0°C in the presence of agonist and decayed by ∼3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at 30°C. The data suggest that a complex of agonist, receptor, and Gs that is primed for the rapid binding of guanine nucleotide can form and be analyzed in reconstituted vesicles.

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