A2-ADRENERGIC RECEPTOR: RECONSTITUTION AND PURIFICATION

A2-肾上腺素能受体：重构和纯化

• 批准号: 3280394
• 负责人: PAUL C STERNWEIS
• 金额: $ 8.51万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1986-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3280394
• 关键词: adenylate cyclase; biotin; detergents; enzyme reconstitution; guanine nucleotides; hormone regulation /control mechanism; ligands; membrane activity; membrane permeability; phospholipids

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.

提出了分离、重构和纯化的策略 α2-肾上腺素能受体。 这些受体介导抑制 α2-肾上腺素能激动剂的腺苷酸环化酶活性。 最初的 实验将集中于溶解和表征 洗涤剂中的受体将增强用于的程序 重构和纯化。 将蛋白质掺入 磷脂囊泡将有助于测量配体的结合 到受体。 部分纯化应将受体从 腺苷酸环化酶的催化和调节成分。 尝试 重组将强调重建双方的互动 具有调节蛋白的受体，据信可以介导 在存在下观察到的腺苷酸环化酶的抑制 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) 与介导的调节蛋白不同 刺激反应，并以纯化形式提供。 标准 受体的成功重建将是：1）关联 以类似于在天然中观察到的方式将配体与受体结合 膜，2）观察α2-肾上腺素能激动剂对 鸟嘌呤核苷酸与胃肠道的关系以及鸟嘌呤的作用 激动剂与受体关联的核苷酸，3) α-肾上腺素能恢复对腺苷酸环化酶活性的抑制 激动剂。 纯化这些受体的尝试将包括 开发使用胃肠道蛋白作为特异性探针的技术。 G 蛋白或其他分子（例如亲和素）的抗体具有高 对附着在 G 蛋白上的化学基团（例如生物素）的亲和力可以 用于特异性分离 GI 和受体的复合物。 旨在解析和重建 Alpha2 肾上腺素能的实验 受体将产生有关激素抑制机制的信息 腺苷酸环化酶。 为重建和开发而开发的技术 该受体的纯化有望应用于其他受体 抑制或激活这种重要调节酶的受体。