BIOCHEMICAL STUDIES OF MUSCARINE RECEPTORS

这项研究计划是专门设计来纯化和表征 毒蕈碱受体来自两个不同的脑区，前脑和 脑干 初步研究表明，毒蕈碱受体 可通过亲和层析在柱上纯化， 琼脂糖-centrine用卡巴胆碱洗脱。 当前应用程序是一个 建议在大鼠中完善后扩大纯化程序 脑组织，以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;确定活性亚基和 通过SDS凝胶电泳测定受体的亚基组成;和 自动微量分析法测定氨基酸组成 通过微丹酰化的N-末端氨基酸。 分离的受体的比较 将使我们能够回答这样一个问题： 这两个区域中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 关于衰老过程和相关的老年痴呆症 疾病影响大脑区域，是有限的。 然而，很明显， 在胆碱能神经传递中存在显著的损伤， 仅限于前脑和海马体，突触后 受体可能是功能障碍的一个部位。 这些研究的结果 可以直接应用于治疗的设计， 在特定的大脑区域，有缺陷的受体。

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.