Ruffolo R R, Fowble J W, Miller D D, Patil P N
Proc Natl Acad Sci U S A. 1976 Aug;73(8):2730-4. doi: 10.1073/pnas.73.8.2730.
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.
强效α-肾上腺素能受体阻断剂阿扎哌汀已用氚气进行催化还原，生成[3H]二氢阿扎哌汀。[3H]二氢阿扎哌汀仍具有显著的阻断α-肾上腺素能受体的能力，并已被用作配体，用于研究含有大鼠输精管膜碎片的亚细胞组分中的受体。[3H]二氢阿扎哌汀的特异性结合迅速达到平衡，也是可逆的且具有饱和性，其解离常数与药理学测定的相似。结合容量约为40 pmol/mg蛋白质。所有测试的α-肾上腺素能阻断剂都能够抑制特异性结合。高浓度的阿普洛尔、阿托品或氯苯那敏没有作用。此外，所有咪唑啉类α-肾上腺素能激动剂在低浓度时均抑制结合，而索特诺尔或氨甲酰胆碱则无此作用。完整组织中受体的阻断或刺激与[3H]二氢阿扎哌汀与亚细胞组分结合的抑制之间存在良好的相关性（r = 0.84）。这些发现表明该组分含有α-肾上腺素能受体相关蛋白。与去甲肾上腺素结构相关的α-肾上腺素能激动剂导致结合呈立体选择性增加，有利于（-）-异构体，这可能反映了受体蛋白上不同结合位点的变构相互作用。提示了结构不同的激动剂存在两种不同结合模式的可能性。
