A2-ADRENERGIC RECEPTOR: RECONSTITUTION AND PURIFICATION

提出了分离、重组和纯化的策略 肾上腺素能受体。 这些受体介导抑制 腺苷酸环化酶活性的α 2肾上腺素能激动剂。 初始 实验将集中在溶解和表征的 洗涤剂中的受体，这将增强用于 重构和纯化。 将蛋白质掺入 磷脂囊泡将促进配体结合的测量 到受体。 部分纯化应将受体从 腺苷酸环化酶的催化和调节组分。 尝试 重建将强调重建的相互作用， 受体与调节蛋白，被认为是介导 腺苷酸环化酶的抑制作用， α-肾上腺素能激动剂 这种鸟嘌呤核苷酸结合调节 蛋白质（GI）不同于调节蛋白质， 刺激反应，并以纯化形式提供。 标准 受体的成功重建将是：1） 配体与受体以类似于在天然细胞中观察到的方式结合。 2）观察α 2-肾上腺素能激动剂对膜的影响， 鸟嘌呤核苷酸与胃肠道的关系及鸟嘌呤的作用 核苷酸对激动剂与受体结合的影响，和3） α-肾上腺素能受体对腺苷酸环化酶活性抑制的恢复 激动剂 纯化这些受体的尝试将包括 开发使用GI蛋白作为特异性探针的技术。 G蛋白或其他分子的抗体（例如，抗生物素蛋白）， 对附着于G蛋白的化学基团的亲和力（例如，生物素）可以 用于特异性分离GI和受体的复合物。 旨在解决和重建α 2-肾上腺素能 受体将产生关于激素抑制的机制的信息， 腺苷酸环化酶 开发的重组技术和 这种受体的纯化将有望适用于其他 受体，抑制或激活这一重要的调节酶。

Strategies are proposed for the isolation, reconstitution, and purification of Alpha2-adrenergic receptors. These receptors mediate inhibition of adenylate cyclase activity by Alpha2-adrenergic agonists. Initial experiments will focus on solubilization and characterization of the receptors in detergents that will enhance procedures to be used for reconstitution and purification. Incorporation of the protein into phospholipid vesicles will facilitate measurement of the binding of ligands to the receptor. Partial purification should resolve the receptor from the catalytic and regulatory components of adenylate cyclase. Attempts at reconstitution will emphasize the reestablishment of the interaction of the receptor with a regulatory protein that is believed to mediate the inhibition of adenylate cyclase observed in the presence of Alpha-adrenergic agonists. This guanine nucleotide-binding regulatory protein (GI) is distinct from the regulatory protein that mediates stimulatory responses and is available in a purified form. Criteria for successful reconstitution of the receptor will be: 1) association of ligands with the receptor in a fashion similar to that observed in native membranes, 2) observation of effects of Alpha2-adrenergic agonists on the association of guanine nucleotides with GI and effects of guanine nucleotide on the association of agonists with receptors, and 3) restoration of inhibition of adenylate cyclase activity by Alpha-adrenergic agonists. Attempts at purification of these receptors will include the development of techniques that use the GI-protein as a specific probe. Antibodies to the G-protein or other molecules (e.g., avidin) with high affinity for chemical groups attached to the G-protein (e.g., biotin) can be used to isolate complexes of GI and the receptor specifically. Experiments designed to resolve and reconstitute the Alpha2-adrenergic receptor will yield information on the mechanism by which hormones inhibit adenylate cyclase. The techniques developed for reconstitution and purification of this receptor will hopefully be applicable to other receptors that either inhibit or activate this important regulatory enzyme.