一种从暗色斑豹纹电鳐电器官中分离纯化烟碱型乙酰胆碱受体的改进方法。

An improved procedure for the isolation and purification of nicotinic acetylcholine receptor from Torpedo fuscomaculata electric organ.

作者信息

Kapp E A, Whiteley C G

机构信息

Department of Chemistry and Biochemistry, Rhodes University, Grahamstown, Republic of South Africa.

出版信息

Biochim Biophys Acta. 1990 Apr 23;1034(1):29-38. doi: 10.1016/0304-4165(90)90149-q.

DOI:10.1016/0304-4165(90)90149-q

PMID:2328259

Abstract

The nicotinic acetylcholinergic receptor has been isolated and purified from extracts of the electric organ of the fish Torpedo fuscomaculata. The isolation procedure involves (a) a series of purification steps including preparation of membrane fragments, extraction of receptors with non-ionic detergents and chromatofocusing; (b) a novel fluorimetric titration assay. The purified receptor is isolated following a 9-fold purification with an overall yield of 12% and a specific activity of 4027 nM.g-1. Gel electrophoresis in the presence of sodium dodecylsulphate produced only one major band with molecular weight of 44,600 associated with the alpha-subunit. A comparison is made with other established procedures. Affinity chromatography on cobratoxin CNBr-Sepharose CL4B produced a 6.8-fold purification, 5% yield and 2900 nM.g-1 specific activity, while in ion-exchange chromatography on DEAE Sepharose 6B gave a 4.7-fold purification, 3% yield and specific activity of 1988 nM.g-1.

摘要

烟碱型乙酰胆碱受体已从暗色斑电鳐的电器官提取物中分离纯化出来。分离过程包括：（a）一系列纯化步骤，包括膜片段的制备、用非离子去污剂提取受体以及色谱聚焦；（b）一种新型荧光滴定测定法。经过9倍纯化后分离得到纯化的受体，总产率为12%，比活性为4027 nM·g⁻¹。在十二烷基硫酸钠存在下进行凝胶电泳，仅产生一条与α亚基相关的主要条带，分子量为44,600。并与其他既定方法进行了比较。在眼镜蛇毒素CNBr-琼脂糖凝胶CL4B上进行亲和层析，纯化倍数为6.8倍，产率为5%，比活性为2900 nM·g⁻¹，而在DEAE琼脂糖凝胶6B上进行离子交换层析，纯化倍数为4.7倍，产率为3%，比活性为1988 nM·g⁻¹。