Photoaffinity labeling of the porcine brain alpha 2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: identification of the hormone-binding subunit.
Photoaffinity labeling of the porcine brain alpha 2-adrenergic receptor using a radioiodinated arylazide derivative of rauwolscine: identification of the hormone-binding subunit.
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使用萝芙素的放射性碘化芳基叠氮化物衍生物对猪脑 α2-肾上腺素受体进行光亲和标记：鉴定激素结合亚基。
DOI：
10.1073/pnas.83.24.9358
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发表时间：
1986
影响因子：
11.1
通讯作者：
Homcy,CJ
中科院分区：
文献类型：
--
作者：
Lanier,SM;Graham,RM;Hess,HJ;Grodski,A;Repaske,MG;Nunnari,JM;Limbird,LE;Homcy,CJ
α 2-选择性拮抗剂rauwolscine的功能化衍生物形成了光亲和加合物的基础，该加合物可以识别大脑α 2-肾上腺素能受体蛋白的激素结合亚基。萝芙木碱羧酸酯与4-N-叔丁氧羰基氨基苯胺反应，合成了萝芙木碱4-氨基苯基甲酰胺（Rau-AmPC）。将Rau-AmPC放射性碘化，并通过重氮盐中间体转化为芳基叠氮化物衍生物17 α-羟基-20 α-育亨班-16 β-[N-（4-叠氮基-3-[125 I]碘）苯基]甲酰胺（125 I-Rau-AzPC）。125 I-Rau-AzPC作为光不稳定探针的表征采用α 2-肾上腺素能受体，其首先从猪脑膜中溶解，并通过亲和层析利用育亨宾-琼脂糖亲和基质部分纯化。在与125 I-Rau-AzPC孵育的部分纯化的受体制剂中，光解导致通过NaDodSO 4/PAGE和放射自显影测定的主要（Mr，62，000）肽的共价标记。这种肽的标记被α 2-选择性拮抗剂育亨宾和非亚型选择性α-拮抗剂酚妥拉明抑制，但不被α 1-拮抗剂哌唑嗪或β-受体拮抗剂（-）-阿普洛尔抑制。α-肾上腺素能激动剂肾上腺素也以立体选择性方式抑制标记。这些数据表明，光标记的Mr 62，000肽是α 2-肾上腺素能受体蛋白的结合亚基。α 2-肾上腺素能受体的放射性碘标记的光亲和探针的可用性应有助于受体蛋白的进一步结构和生物物理表征。
A functionalized derivative of the alpha 2-selective antagonist rauwolscine formed the basis for a photoaffinity adduct that has allowed identification of the hormone-binding subunit of the brain alpha 2-adrenergic receptor protein. Rauwolscine carboxylate underwent reaction with 4-N-t-butyloxycarbonyl-aminoaniline, leading to the synthesis of rauwolscine 4-aminophenyl carboxamide (Rau-AmPC). Rau-AmPC was radioiodinated and converted to the arylazide derivative, 17 alpha-hydroxy-20 alpha-yohimban-16 beta-[N-(4-azido-3-[125I]iodo)phenyl] carboxamide (125I-Rau-AzPC), via a diazonium salt intermediate. The characterization of 125I-Rau-AzPC as a photolabile probe employed alpha 2-adrenergic receptors, which were first solubilized from porcine brain membranes and partially purified by affinity chromatography utilizing a yohimbine-agarose affinity matrix. In the partially purified receptor preparation incubated with 125I-Rau-AzPC, photolysis resulted in covalent labeling of a major (Mr, 62,000) peptide as determined by NaDodSO4/PAGE and autoradiography. Labeling of this peptide was inhibited by the alpha 2-selective antagonist, yohimbine, and the non-subtype-selective alpha-antagonist, phentolamine, but not by the alpha 1-antagonist, prazosin, or the beta-receptor antagonist, (-)-alprenolol. The alpha-adrenergic agonist epinephrine also inhibited labeling in a stereoselective manner. These data indicate that the photolabeled Mr 62,000 peptide is the hormone-binding subunit of the alpha 2-adrenergic receptor protein. The availability of this radioiodinated photoaffinity probe for the alpha 2-adrenergic receptor should facilitate further structural and biophysical characterization of the receptor protein.