BIOCHEMICAL AND IMMUNOLOGICAL STUDIES OF MUSCURINE RECEP

现有证据支持胆碱能功能降低在糖尿病中的作用 自然衰老过程中的记忆丧失和认知障碍 阿尔茨海默氏症。我们的研究结果显示，M2的选择性损失 突触前胆碱能终末M受体亚型 胆碱乙酰转移酶与晚期阿尔茨海默病的关系 突触后ML亚型。胆碱能疗法治疗慢性阻塞性肺疾病 因此，阿尔茨海默氏症必须针对M1受体 机制。此应用程序的总体目标是定义 用生化和免疫组织化学方法研究M1和M2受体亚型的特性 免疫学技术。拟议的工作将解决一个分歧 在药理学、解剖学和生理学证据之间 两种M受体亚型和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。兔的M受体 海马体和脑干，富含M1和M2(约90%) 亚型将分别溶解在洋地黄素中，并通过 层析技术的结合。受体将被表征为 按大小、等电点、氨基酸组成和 多肽图。巯基和二硫键在配体中的作用 将检查结合和受体构象。单克隆抗体 将从兔的海马区和海马区产生可溶性受体部分 脑干。将对抗体进行筛选，以确定其识别能力 各种形式的受体，如天然的、变性的、溶液中游离的或 固定在塑料上。抗体将被用来确认或否认 两种截然不同的受体蛋白的存在 抗体与两种不同的受体制剂发生交叉反应。 将测试抗体识别受体的能力。 各种形式的蛋白质，即作为纯蛋白质，变性，结合到 配体，或在受体-效应器复合体中。还将使用抗体 用于免疫亲和纯化受体蛋白，可以 进一步研究了生物化学。最后，表现出特异性的抗体 与M1或M2子类型的绑定将用于 这些受体在卵巢癌组织切片中的免疫组织化学定位 兔子和人类的大脑。这些研究将提供有用的信息，如 正常衰老、老年期痴呆和老年性痴呆患者血中M受体的变化 阿尔茨海默氏症。

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.