Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.
Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.
复制标题
通过带正电荷的滤膜上的蛋白质转移分析 α-银环蛇毒素与鱼雷乙酰胆碱受体 α 亚基的蛋白水解片段的结合。
DOI：
10.1073/pnas.81.8.2553
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发表时间：
1984
影响因子：
11.1
通讯作者：
Lentz,TL
中科院分区：
文献类型：
--
作者：
Wilson,PT;Gershoni,JM;Hawrot,E;Lentz,TL
乙酰胆碱受体α亚基的蛋白水解片段在聚丙烯酰胺凝胶电泳和蛋白质转移固定化后保留了与α -班加罗毒素结合的能力。用金黄色葡萄球菌V-8蛋白酶、木瓜蛋白酶、菠萝蛋白酶、k蛋白酶四种蛋白酶对鱼雷电器官乙酰胆碱受体α亚基进行酶解，将15%聚丙烯酰胺凝胶分解的蛋白片段电泳转移到带正电的尼龙膜过滤器上。当与0.3 nM 125i标记的α -班加罗毒素孵育并进行放射自显影时，转移产生了每种蛋白酶特有的标记带模式。结合毒素片段的分子量范围为7 ~ 34 kDa，主要分布在8-、18-和28-kDa。IC50值为6.7 X 10(-8) m。含有受体的膜与4-（n -马来酰亚胺）- α -苄基三甲基碘化铵的预先亲和烷基化可以抑制α -班加罗毒素片段的标记。这些发现表明，小至α亚基大小的1/5的固定化蛋白水解片段保留了结合α -班加罗毒素所必需的结构特征，尽管毒素与片段的结合比与天然受体的亲和力低。亲和配体烷基化的影响表明，在蛋白水解片段上检测到的-班加罗毒素结合位点与完整乙酰胆碱受体上亲和标记的乙酰胆碱结合位点相同。
Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.