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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1976 Aug;73(8):2730–2734. doi: 10.1073/pnas.73.8.2730

Binding of [3H]dihydroazapetine to alpha-adrenoreceptor-related proteins from rat vas deferens.

R R Ruffolo Jr, J W Fowble, D D Miller, P N Patil
PMCID: PMC430722  PMID: 8779

Abstract

The potent alpha-adrenoreceptor blocking agent, azapetine, has been catalytically reduced with tritium gas to form [3H]dihydroazapetine. [3H]Dihydroazapetine retains significant ability to block alpha-adrenoreceptors and has been used as a ligand to study the receptor in a subcellular fraction containing membrane fragments from rat vas deferens. Specific binding of [3H]dihydroazapetine rapidly reaches equilibrium and is also reversible and saturable with a dissociation constant similar to that determined pharmacologically. The binding capacity is approximately 40 pmol/mg of protein. All alpha-adrenergic blockers tested were able to inhibit specific binding. High concentrations of alprenolol, atropine, or chlorpheniramine had no effect. In addition, all alpha-adrenergic agonists of the imidazoline class inhibit binding in low concentrations, whereas soterenol or carbamylcholine did not. There is good correlation (r=0.84) between blockade or stimulation of the receptor in intact tissues and inhibition of binding of [3H]dihydroazapetine to the subcellular fraction. These findings suggest that the fraction contains alpha-adrenoreceptor-related proteins. Alpha-adrenergic agonists structurally related to norepinephrine caused a stereoselective increase in binding in favor of the (-)-isomer, possibly reflecting an allosteric interaction at a different binding site on the receptor protein. The possibility of two different modes of binding for structurally dissimilar agonists is suggested.

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Selected References

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