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

为了研究受体控制膜透性机制的分子基础，需要获得相当数量的高纯度受体。尽管人们认为烟碱乙酰胆碱受体（nAChR）在大多数组织中浓度极低，但电鱼器官提供了相对丰富的受体来源。结果：主要结果总结如下：采用cobrotoxin-Sepharose偶联物作为吸附剂进行亲和层析，从加利福尼亚鱼雷（Torpedo californica）的电器官中高度纯化了nAChR。对纯化后的nAChR的碳水化合物结构进行了分析。nAChR的低聚糖链中70%以上为高甘露糖型，结构为Man_8GlcNAc_2和Man_9GlcNAc_2。这些低聚糖以不同的比例分布在所有亚基中。其余寡糖为多种复合类型，主要存在于< γ >和< δ >亚基。此外，对碳水化合物部分特别是nAChR配体结合的功能作用的研究表明，至少在成熟的nAChR中，nAChR的碳水化合物部分在胆碱能配体的结合中不是必需的。从加利福尼亚鱼电器官的nachr富集膜中，通过色谱法可纯化出蛋白激酶（<nu>_2）和肌酸激酶（<nu>_1）等分子量为43000个蛋白（43K），均质化。在外源性测定系统中发现，该蛋白激酶磷酸化nAChR，其氨基末端序列与从各种动物中分离的肌酸激酶具有极高的同源性。虽然nAChR的分子性质已经得到了相当的证实，但nAChR的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

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