Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.
Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.
复制标题
α-银环蛇毒素与加州鱼雷乙酰胆碱受体分离的 α 亚基的结合：蛋白质印迹定量分析。
DOI：
10.1073/pnas.80.16.4973
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发表时间：
1983
影响因子：
11.1
通讯作者：
Lentz,TL
中科院分区：
文献类型：
--
作者：
Gershoni,JM;Hawrot,E;Lentz,TL
The direct binding of alpha-bungarotoxin to the alpha subunit of the acetylcholine receptor from Torpedo electric organ immobilized onto protein blots was demonstrated. Protein blots were prepared by electrophoretically transferring resolved acetylcholine receptor subunits from 10% polyacrylamide gels onto Zetabind, positively charged nylon membrane filters. Such blots, when incubated with 125I-labeled alpha-bungarotoxin, washed, and autoradiographed, gave rise to a single labeled band corresponding to the alpha subunit of the receptor. The labeling with alpha-bungarotoxin could be inhibited by pretreating the receptor-containing membranes with the affinity ligand 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. In addition, the association of toxin with the alpha subunit could be inhibited by d-tubocurarine (IC50 = 0.9 mM). Furthermore, removal of high-mannose oligosaccharide chains from the alpha subunit by treatment with endoglycosidase H did not interfere with the observed toxin binding. Thus it is demonstrated that isolated, immobilized alpha subunit of the Torpedo acetylcholine receptor can bind alpha-bungarotoxin. However, the observed binding of alpha-bungarotoxin to immobilized alpha subunit is reduced in affinity to 1/1,000 to 1/10,000 of that obtained with native receptor. The endoglycosidase H-susceptible oligosaccharide side chain(s) is not required for this interaction. Binding of alpha-bungarotoxin is to the physiologically relevant acetylcholine binding site as defined by affinity ligand alkylation.
