Lindstrom J, Anholt R, Einarson B, Engel A, Osame M, Montal M
J Biol Chem. 1980 Sep 10;255(17):8340-50.
We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubalization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naga naja siamensis toxin III or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamycholine-induced influx of 22Na+ per mol of receptor. Carbamylcholine-induced 22Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles 22Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.
我们报告了通过五种方法从加州电鳐电器官组织中纯化具有活性激动剂调节阳离子通道的乙酰胆碱受体。在一种其他人先前使用过的方法中，通过碱性提取从部分纯化的膜中去除污染蛋白，在整个过程中保持膜的完整性。在其他四种方法中，用胆酸钠溶解后纯化乙酰胆碱受体。胆酸盐混合胶束中持续存在大豆脂质是防止阳离子通道不可逆失活所必需的。溶解的受体通过使用与琼脂糖偶联的眼镜蛇毒素III或伴刀豆球蛋白A进行亲和层析来纯化。蔗糖梯度离心也用于纯化溶解的受体。最佳方法是将毒素琼脂糖亲和层析和伴刀豆球蛋白A琼脂糖亲和层析结合起来。通过所有五种方法纯化的受体通过胆酸盐透析技术整合到大豆脂质小泡中。受体的激动剂调节阳离子通道在重构后同样具有活性，与用于纯化的方法无关。所有重构的小泡制剂在乙酰胆碱受体对外表面的优先取向、对氨甲酰胆碱的剂量反应、氨甲酰胆碱诱导的脱敏以及每摩尔受体氨甲酰胆碱诱导的22Na+内流方面都相似。氨甲酰胆碱诱导的22Na+内流/受体在重构后比在天然小泡中更大。这是因为，在天然小泡中，氨甲酰胆碱诱导的22Na+内流受小泡内部体积与外部22Na+浓度平衡的限制，而在重构小泡中，22Na+内流受受体分子脱敏的限制。我们证明，受体单体上的两个毒素结合位点中只有一个，即可以用4-(N-马来酰亚胺基)苄基三甲基铵进行亲和烷基化的那个位点，控制氨甲酰胆碱诱导的阳离子通道开放。
