The acetylcholine receptor. I. Purification and characterization of a macromolecule isolated from Electrophorus electricus.
The acetylcholine receptor. I. Purification and characterization of a macromolecule isolated from Electrophorus electricus.
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乙酰胆碱受体。
DOI：
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发表时间：
1973
影响因子：
4.8
通讯作者：
L. Possani
中科院分区：
文献类型：
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作者：
R. Klett;B. Fulpius;D. Cooper;M. Smith;E. Reich;L. Possani
摘要 从鳗鱼电器官的提取物中分离出烟碱乙酰胆碱受体。分离程序涉及以下要素：（a）基于受体结合眼镜蛇-[3H]神经毒素的能力的快速且可重复的测定，并允许使用 DEAE-纤维素纸盘测量复合物形成； (b)一系列纯化步骤，包括制备膜片段、用非离子去污剂提取受体，然后进行凝胶渗透、亲和和离子交换层析。经过 8250 倍纯化后分离出纯化的受体，总收率约为 5%。该受体是一种大蛋白质，不含吸收紫外线的辅基，在天然和变性时均在聚丙烯酰胺凝胶电泳中作为单条带迁移。氨基酸分析将该蛋白质与乙酰胆碱酯酶区分开来。最好的制剂每 90,000 克蛋白质可结合 1 摩尔神经毒素，并且形成复合物的能力对热和蛋白水解敏感。分子量和亚基结构仍有待确定。受体与眼镜蛇神经毒素之间的反应缓慢地向平衡状态进行。受体对毒素的表观亲和力随着孵育时间的延长而增加。毒素受体平衡随着温度的变化而变化，这种变化完全是由于缔合速率的变化，解离速率仍然与温度无关。 25° 时的动力学和热力学参数为：k1 = 1.35 x 105 摩尔-1 s-1，k-1 = 1.52 x 10-4 s -1，Kd = 1.13 x 10-9 m，ΔH0 = 15.3 卡每摩尔，ΔG0 = -12.2 卡每摩尔，ΔS0 = +92 e.u。毒素-受体平衡和 DEAE 滤纸测定为研究受体与胆碱能配体的相互作用提供了一种方便的方法。
Abstract The nicotinic acetylcholine receptor has been isolated from extracts of the electric organ of the eel, Electrophorus electricus. The isolation procedure involves the following elements: (a) a rapid and reproducible assay that is based on the ability of receptor to bind cobra-[3H] neurotoxin and allows complex formation to be measured using DEAE-cellulose paper discs; (b) a series of purification steps including preparation of membrane fragments, extraction of receptors with nonionic detergents, followed by gel permeation, affinity and ion exchange chromatography. The purified receptor is isolated following 8250-fold purification with an over-all yield of approximately 5%. The receptor is a large protein that is free of ultraviolet absorbing prosthetic groups, and migrates as a single band in polyacrylamide gel electrophoresis both when native and denatured. The amino acid analysis distinguishes this protein from acetylcholinesterase. The best preparations bind 1 mole of neurotoxin per 90,000 g of protein, and the ability to form complexes is sensitive to heat and proteolysis. The molecular weight and subunit structure remain to be determined. The reaction between receptor and cobra neurotoxin proceeds toward the equilibrium state slowly. The apparent affinity of receptor for toxin increases with longer times of incubation. The toxin receptor equilibrium changes as a function of temperature, and this change is entirely due to changes in the rate of association, the dissociation rate remaining temperature independent. The kinetic and thermodynamic parameters at 25° are: k1 = 1.35 x 105 mole-1 s-1, k-1 = 1.52 x 10-4 s -1, Kd = 1.13 x 10-9 m, ΔH0 = 15.3 Cal per mole, ΔG0 = -12.2 Cal per mole, ΔS0 = +92 e.u. The toxin-receptor equilibrium and the DEAE-filter paper assay provide a convenient method for studying the interaction of receptor with cholinergic ligands.