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

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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