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Biochemical Studies on the Mechanism of Neurotransmission--- Principally Structure-Function Relationship of Nicotinic Acetylcholine Receptor ---

神经传递机制的生化研究---主要是烟碱乙酰胆碱受体的结构与功能关系---

基本信息

批准号：
61440085
负责人：
HAYASHI Kyozo
金额：
$ 18.69万
依托单位：
Gifu Pharmaceutical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

In order to study the molecular basis of the mechanism by which receptors control membrane permeability, it is required to obtain a considerable of the highly purified receptor. Although nicotinic acetylcholine receptor (nAChR) is thought to be present in extremely low concentration in most tissues, electric fish organs provide a relatively rich source of the receptor.Results: Major results are summarized as follows;1. nAChR was highly purified from the electric organ of Torpedo californica, by employing a cobrotoxin-Sepharose conjugate as an adsorbent for affinity chromatography.2. The carbohydrate structures of the purified nAChR was eluciadated. More than 70% of the total oligosaccharide chain of nAChR are of the high-mannose type with the structures Man_8GlcNAc_2 and Man_9GlcNAc_2. These oligosaccharides distributed among all subunits at different proportions. The remaining oligosaccharides were several complex types, and the type existed mainly in <gamma> and <delta> subunits.3. T … More he examination of the functional role of the carbohydrate moiety especially in ligand-binding of nAChR revealed that the carbohydrate moiety of nAChR is not necessary in the binding of cholinergic ligands at least in matured form of nAChR.4. The molecular weight 43,000 proteins (43K) such as the protein kinase (<nu>_2) and creatine kinase (<nu>_1) could be purified from the nAChR-enriched membranes of the electric organ of Torpedo californica to homogeneity by chromatographic procedures. It was found that the protein kinase phosphorylates the nAChR in an exogenous assay system and the amino-terminal sequence of the creatine kinase shows the extremely high homology with those of creatine kinases isolated from various animals.Although the molecular natures of nAChR have been fairly demonstrated, the detailed mechanism of the neurotransmission of nAChR still remains to be clarified. The elucidation of the three-dimentional structure of nAChR by X-ray analysis will open the way to a better under-standing of the problem. Less

为了研究受体控制膜通透性的机制的分子基础，需要获得相当数量的高度纯化的受体。虽然烟碱型乙酰胆碱受体（nAChR）被认为在大多数组织中以极低的浓度存在，但电鱼器官提供了相对丰富的受体来源。采用cobrotoxin-Sepharose偶联物作为亲和层析吸附剂，从加州电鳐（Torpedocalfornica）的电器官中高效纯化了nAChR.对纯化的nAChR的糖结构进行了解析。超过70%的nAChR的总寡糖链是具有结构Man_8GlcNAc_2和Man_9GlcNAc_2的高甘露糖型。这些寡糖以不同的比例分布在各亚基之间。剩余的低聚糖是几种复杂的类型，主要存在于<gamma>和<delta>亚基中.不 ...更多信息碳水化合物部分的功能作用的检查，特别是在nAChR的配体结合中，揭示了nAChR的碳水化合物部分在胆碱能配体的结合中不是必需的，至少在成熟形式的nAChR中是如此。从加州电鳐（Torpedocalfornica）电器官的nAChR富集膜中可纯化出分子量为43，000（43 K）的蛋白激酶（<nu>_2）和肌酸激酶（<nu>_1）。在外源性检测系统中发现，该蛋白激酶可使nAChR磷酸化，其氨基端序列与多种动物肌酸激酶的氨基酸序列具有很高的同源性，虽然nAChR的分子性质已得到充分的证实，但其神经传递的详细机制仍有待阐明。通过X射线分析阐明nAChR的三维结构将为更好地理解这一问题开辟道路。少