Purification and characterization of a nicotinic acetylcholine receptor from chick brain.
Purification and characterization of a nicotinic acetylcholine receptor from chick brain.
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DOI：
10.1021/bi00356a037
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发表时间：
1986-04
期刊：
影响因子：
2.9
通讯作者：
P. Whiting;J. Lindstrom
中科院分区：
文献类型：
--
作者：
P. Whiting;J. Lindstrom
免疫组织化学研究表明，鸡脑和鸡睫状神经节神经元含有一个组件，该组件与电气器官和骨骼肌的烟碱乙酰胆碱受体的主要免疫原性区域共享抗原决定簇。在这里，我们描述了这种假定的神经乙酰胆碱受体的纯化和初步表征。通过单克隆抗体亲和层析纯化组分。溶解的成分沉淀在蔗糖梯度作为一个物种略大于电鳐乙酰胆碱受体单体。用溴[3 H]乙酰胆碱进行亲和标记。标记被卡巴胆碱阻止，但不是由α-银环蛇毒素。在亲和纯化的组分中可以检测到两个亚基，表观分子量为48 000和59 000。分子量为48000的亚基被针对电器官和骨骼肌乙酰胆碱受体的主要免疫原性区域的单克隆抗体和针对电鳐受体α亚基的抗血清结合。有证据表明，大脑中有两个α亚基。用纯化的脑成分免疫的大鼠的抗血清与电鳐电器官或鸡肌肉乙酰胆碱受体表现出很少或没有交叉反应。然而，一种抗血清确实特异性地结合鱼雷受体的所有四个亚基。实验将在别处描述（J.斯托尔贝格等人，未公开的结果）显示，纯化的脑组分的抗血清特异性抑制鸡睫状神经节神经元中乙酰胆碱受体的电生理功能，而不抑制鸡肌肉细胞中乙酰胆碱受体的功能。所有这些特性表明，该组分是一种神经元烟碱乙酰胆碱受体，与肌肉烟碱乙酰胆碱受体具有有限的结构同源性。
Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.