加州电鳐乙酰胆碱受体α链中乙酰胆碱结合位点的定位与合成
Localization and synthesis of the acetylcholine-binding site in the alpha-chain of the Torpedo californica acetylcholine receptor.
作者信息
McCormick D J, Atassi M Z
出版信息
Biochem J. 1984 Dec 15;224(3):995-1000. doi: 10.1042/bj2240995.
Abstract
The sequence of the alpha-chain of the acetylcholine receptor of T. californica has been determined by recent cloning studies. The integrity of the disulphide bond between Cys-128 and cys-142 has been shown to be important for the maintenance of the binding activity of the receptor, thus implicating the regions around the disulphide bridge in binding with acetylcholine. In the present work, a synthetic peptide containing this loop region (residues 125-147) was synthesized. Solid-phase radiometric binding assays demonstrated a high binding of 125I-labelled alpha-bungarotoxin to the synthetic peptide. It was further shown that the free peptide bound well to [3H]acetylcholine. Additional experiments demonstrated that pretreatment of peptide 125-147 with 2-mercaptoethanol destroyed its binding activity, clearly showing that the integrity of the disulphide structure was essential for binding. Unlabelled acetylcholine also inhibited the binding of labelled acetylcholine to the synthetic peptide. The region 125-147, therefore, contains essential elements of the acetylcholine binding site of the Torpedo receptor.
摘要
近期的克隆研究已确定了加州电鳐乙酰胆碱受体α链的序列。已证明半胱氨酸-128与半胱氨酸-142之间二硫键的完整性对于维持受体的结合活性很重要，因此表明二硫桥周围区域参与与乙酰胆碱的结合。在本研究中，合成了包含该环区（第125 - 147位氨基酸残基）的合成肽。固相放射性结合试验表明，125I标记的α-银环蛇毒素与合成肽有高度结合。进一步表明游离肽与[3H]乙酰胆碱结合良好。额外实验证明，用2-巯基乙醇预处理肽125 - 147会破坏其结合活性，清楚地表明二硫键结构的完整性对于结合至关重要。未标记的乙酰胆碱也抑制标记的乙酰胆碱与合成肽的结合。因此，第125 - 147位区域包含了电鳐受体乙酰胆碱结合位点的关键元件。
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High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate.α-银环蛇毒素与纯化的α亚基及其来自电鳐乙酰胆碱受体的27000道尔顿蛋白水解肽的高亲和力结合。对十二烷基硫酸钠的需求。
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