Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.
Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.
复制标题
确定加州鱼雷乙酰胆碱受体 α 亚基上 α-银环蛇毒素结合位点的一级氨基酸序列。
DOI：
10.1073/pnas.82.24.8790
复制
发表时间：
1985
影响因子：
11.1
通讯作者：
Hawrot,E
中科院分区：
文献类型：
--
作者：
Wilson,PT;Lentz,TL;Hawrot,E
烟碱乙酰胆碱受体α亚基含有α -班加罗毒素结合域的区域被映射到与天冬酰胺-141相关的初级氨基酸序列上，天冬酰胺-141是假定的n链糖基化位点。α亚基的蛋白水解片段固定在带正电的膜过滤器上，根据片段的大小和对h内糖苷酶消化的敏感性测定的天冬酰胺-141的存在，进一步分析了与125i标记的班加罗毒素结合的片段。发现班加罗毒素结合位点不在氨基酸残基1和140之间，因为检测到班加罗毒素结合片段比140个氨基酸大得多，并且缺乏n连接的低聚糖链。含有天冬酰胺-141的最小的邦加罗毒素结合片段的大小和V8蛋白酶酶切产生的片段的大小进一步表明邦加罗毒素结合位点位于氨基酸残基153-241内。一个32个氨基酸的合成肽包含该区域的一部分（残基173-204）被测试其结合125i标记的班加罗毒素的能力。125i标记的班加罗毒素与肽结合，与未标记的班加罗毒素和d-班加罗毒素竞争，IC50值分别为0.5微米和2毫米。我们得出结论，在α亚基上的虫毒结合位点的主要决定因素位于残基173和204之间。
A region of the alpha subunit of the nicotinic acetylcholine receptor containing the alpha-bungarotoxin-binding domain was mapped on the primary amino acid sequence in relation to asparagine-141, the presumed site of N-linked glycosylation. Proteolytic fragments of the alpha subunit, immobilized onto positively charged membrane filters, that bind 125I-labeled bungarotoxin were further analyzed on the basis of the size of the fragments and the presence of asparagine-141 as determined by susceptibility to digestion with endoglycosidase H. The bungarotoxin-binding site was found not to reside between amino acid residues 1 and 140 since bungarotoxin-binding fragments that are considerably larger than 140 amino acids and lack N-linked oligosaccharide chains were detected. The size of the smallest bungarotoxin-binding fragment containing asparagine-141 and the size of fragments produced by digestion with V8 protease further indicated that the bungarotoxin-binding site is contained within amino acid residues 153-241. A 32-amino acid synthetic peptide comprising a portion of this region (residues 173-204) was tested for its ability to bind 125I-labeled bungarotoxin. 125I-labeled bungarotoxin bound to the peptide and was competed by unlabeled bungarotoxin and d-tubocurarine with IC50 values of 0.5 microM and 2 mM, respectively. We conclude that a major determinant of the bungarotoxin-binding site on the alpha subunit resides between residues 173 and 204.