Cornett L E, Norris J S
Biochem Pharmacol. 1986 May 15;35(10):1663-9. doi: 10.1016/0006-2952(86)90320-5.
In this study, we used phenoxybenzamine to label the alpha 1-adrenergic receptor of a smooth muscle cell line. Our results demonstrate a dose-dependent occupancy of alpha 1-adrenergic receptors by phenoxybenzamine determined by competition for the [3H]prazosin binding site. Following incorporation of [3H]phenoxybenzamine, partially purified membranes were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Despite numerous Coomassie blue-stained bands, only three bands, Mr = 80,000 +/- 500, Mr = 33,000 +/- 2,000, and Mr = 21,000 +/- 400 (N = 4), were labeled with [3H]phenoxybenzamine as determined by autofluorography. Incorporation of [3H]phenoxybenzamine into the Mr = 80,000 band, but not the Mr = 33,000 and Mr = 21,000 bands, was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Labeling of the Mr = 33,000 and Mr = 21,000 bands was partially blocked by phenoxybenzamine. We conclude that [3H]phenoxybenzamine can be used as an affinity probe for the alpha 1-adrenergic receptor and that the ligand binding site of the alpha 1-adrenergic receptor resides in a Mr = 80,000 protein.
在本研究中，我们使用酚苄明标记一种平滑肌细胞系的α1-肾上腺素能受体。我们的结果表明，通过对[3H]哌唑嗪结合位点的竞争测定，酚苄明对α1-肾上腺素能受体的占据呈剂量依赖性。掺入[3H]酚苄明后，将部分纯化的膜溶解，并在还原条件下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳。尽管考马斯亮蓝染色带众多，但通过自动放射自显影测定，只有三条带被[3H]酚苄明标记，其分子量分别为Mr = 80,000 +/- 500、Mr = 33,000 +/- 2,000和Mr = 21,000 +/- 400（N = 4）。肾上腺素能激动剂和拮抗剂对[3H]酚苄明掺入Mr = 80,000的条带（而非Mr = 33,000和Mr = 21,000的条带）有影响，其方式与α1-肾上腺素能相互作用一致。酚苄明可部分阻断Mr = 33,000和Mr = 21,000条带的标记。我们得出结论，[3H]酚苄明可作为α1-肾上腺素能受体的亲和探针，且α1-肾上腺素能受体的配体结合位点存在于一种分子量为Mr = 80,000的蛋白质中。
