BIOCHEMICAL STUDIES OF MUSCARINE RECEPTORS

毒蕈碱受体的生物化学研究

• 批准号: 3449655
• 负责人: DONNA D FLYNN
• 金额: $ 5.65万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-02-01 至 1986-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3449655
• 关键词: affinity chromatography; autoradiography; brain stem; electrofocusing; gel electrophoresis; gel filtration chromatography; ion exchange chromatography; neural transmission; neurochemistry; prosencephalon

This research proposal is specifically designed to purify and characterize the muscarine receptor from two separate brain regions, forebrain and brainstem. Preliminary work has demonstrated that the muscarine receptor is amenable to purification by affinity chromatography on columns of Sepharose-centrine eluted with carbachol. The current application is a proposal to scale-up the purification procedures, after perfection in rat brain tissue, to work with 60 g. lots of bovine brain tissue, which have about 1. mg of pure receptor. Characterization of the purified receptor will include: measurement of molecular weight by sedimentation equilibrium or sedimentation velocity; determination of the active subunit and the subunit composition of the receptor by SDS gel electrophoresis; and determination of amino acid composition by automated microanalysis and the N-terminal amino acid by microdansylation. Comparison of receptor isolated from these brain regions will enable us to answer the question of whether differences in agonist and antagonist binding in these two regions are due to structurally different receptors. This question is significant because current knowledge, of how the aging process and the related Alzheimer's disease affects brain regions, is limited. It is apparent, however, that there is a significant impairment in cholinergic neurotransmission, confined to only the forebrain and hippocampus, and that the postsynaptic receptor may be a site of the dysfunction. The results of these studies can be directly applied to the design of therapies to specifically target receptors, in a particular brain region, that are defective.

该研究计划专门设计用于纯化和表征 来自两个独立大脑区域的毒蕈碱受体，前脑和 脑干。 初步工作表明毒蕈碱受体 适合通过亲和层析柱进行纯化 用卡巴胆碱洗脱琼脂糖中心素。 当前的应用程序是一个 建议在大鼠中完善后扩大纯化程序 脑组织，使用 60 g。大量的牛脑组织， 约1.mg纯受体。 纯化受体的表征 将包括： 通过沉降平衡测量分子量 或沉降速度；活性亚基的测定和 通过SDS凝胶电泳检测受体的亚基组成；和 通过自动微量分析测定氨基酸组成 N-末端氨基酸通过微丹酰化。 分离受体的比较 从这些大脑区域中我们可以回答以下问题： 这两个区域激动剂和拮抗剂结合的差异是由于 结构不同的受体。 这个问题很重要，因为 当前关于衰老过程和相关阿尔茨海默病的知识 疾病对大脑区域的影响是有限的。 然而，显然， 胆碱能神经传递显着受损， 仅限于前脑和海马体，突触后 受体可能是功能障碍的部位。 这些研究的结果 可以直接应用于针对特定目标的疗法设计 特定大脑区域的受体有缺陷。