Rapid binding of guanosine 5'-O-(3-thiotriphosphate) to an apparent complex of beta-adrenergic receptor and the GTP-binding regulatory protein Gs.
When reconstituted phospholipid vesicles that contain purified beta-adrenergic receptors and the GTP-binding regulatory protein Gs were preincubated with agonist before the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), the typical receptor-stimulated GTP gamma S binding reaction was preceded by an even more rapid burst of GTP gamma S binding. This burst was studied in detail at 0 degree C. The rate of the burst was second order in nucleotide and Gs [k assoc approximately 2 X 10(7) (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 approximately 15 min at 0 degree C in the presence of agonist and decayed by approximately 3 min upon addition of antagonist or detergent. Formation and decay of this species was much faster at at 30 degrees 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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Department of Pharmacology, Southwestern Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Ross
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D000818:Animals; D004912:Erythrocytes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D011485:Protein Binding; D011817:Rabbits; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*; D014422:Turkeys
D046911:Macromolecular Substances; D011943:Receptors, Adrenergic, beta; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins