BIOCHEMICAL AND IMMUNO STUDIES OF MUSCARINE RECEP

现有证据支持胆碱能功能降低在 自然衰老过程中的记忆丧失和认知障碍， 老年痴呆症 我们的研究结果表明，M2的选择性损失 突触前胆碱能末梢上的毒蕈碱受体亚型沿着 胆碱乙酰转移酶在晚期阿尔茨海默病和 突触后M1亚型。 胆碱能疗法用于治疗 因此，阿尔茨海默氏症痴呆症必须针对M1受体 机制 此应用程序的总体目标是定义 M1和M2受体亚型的特性， 免疫技术。 拟议的工作将解决一个矛盾 在药理学解剖学和生理学证据之间 两种毒蕈碱受体亚型，以及一种常见的生化证据， 受体蛋白亚基，通过确认或否认存在 不同的（完整的）受体蛋白。 家兔毒蕈碱受体 海马和脑干，富含（约90%）M1和M2 亚型分别溶解在毛地黄皂苷中，并通过 色谱技术的组合。 将表征受体 在生物化学上，通过大小、等电点、氨基酸组成和 肽图谱 巯基和二硫基在配体中的作用 将检查结合和受体构象。 单克隆抗体 将产生来自兔海马的可溶性受体级分， 脑干 将筛选抗体识别 各种形式的受体，例如天然的、变性的、游离于溶液中的或 固定在塑料上。 抗体将用于确认或否认 存在两种不同的受体蛋白的能力， 抗体与两种不同的受体制剂交叉反应。 将测试抗体识别受体的能力 各种形式的蛋白质，即纯蛋白质，变性的，结合的， 配体或受体-效应物复合物中。 抗体也将被用于 用于受体蛋白的免疫亲和纯化， 进一步的生物化学研究。 最后，显示出特异性的抗体 与M1或M2亚型结合的能力将用于 这些受体在组织切片中的免疫组织化学定位 兔子和人脑 这些研究将提供有用的信息， 正常衰老、老年性痴呆和 老年痴呆症

Available evidence supports a role for decreased cholinergic function in memory loss and cognitive impairments in the natural aging process and Alzheimer's disease. Our findings show a selective loss of the M2 muscarine receptor subtype on presynaptic cholinergic terminals along with choline acetyltransferase in advanced Alzheimer's disease and retention of the postsynaptic Ml subtype. Cholinergic therapy for the treatment of Alzheimer's dementia must therefore be directed at the M1 receptor mechanism. The overall goal of this application is to define the properties of M1 and M2 receptor subtypes using biochemical and immunological techniques. The proposed work will resolve a discrepancy between pharmacological, anatomical and physiological evidence in favor of two muscarine receptor subtypes, and biochemical evidence for one common receptor protein subunit, by confirming or denying the existence of distinct (whole) receptor proteins. Muscarine receptors from rabbit hippocampus and brainstem, which are rich (approximately 90%) in M1 and M2 subtypes, respectively, will be solubilized in digitonin and purified by a combination of chromatographic techniques. Receptors will be characterized biochemically by size, isoelectric point, amino acid composition, and peptide maps. The role of sulfhydryl and disulfide groups in ligand binding and receptor conformation will be examined. Monoclonal antibodies will be produced to soluble receptor fractions from rabbit hippocampus and brainstem. Antibodies will be screened for their ability to recognize receptors in various forms such as native, denatured, free in solution or immobilized on plastic. Antibodies will be used to confirm or deny the existence of two distinct receptor proteins by the ability of the antibodies to cross-react with the two distinct receptor preparations. Antibodies will be tested for their ability to recognize the receptor proteins in various forms, i.e. as pure proteins, denatured, bound to ligands, or in receptor-effector complexes. Antibodies will also be used for immunoaffinity purification of the receptor proteins which can be further studied biochemically. Finally, antibodies that show specificity of binding to either the M1 or M2 subtypes will be used for immunohistochemical localization of these receptors in tissue sections from rabbit and human brains. These studies will provide useful information on changes in muscarine receptors in normal aging, senile dementia and Alzheimer's disease.