Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.
Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.
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合成肽的抗体作为乙酰胆碱受体 α 亚基上结合位点的探针。
DOI：
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发表时间：
1985
影响因子：
11.1
通讯作者：
S. Fuchs
中科院分区：
文献类型：
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作者：
D. Neumann;J. Gershoni;M. Fridkin;S. Fuchs
使用合成肽及其各自的抗体来尝试定位和鉴定烟碱乙酰胆碱受体的配体结合位点。合成了受体α亚基的两个肽，第一个对应于NH2末端结构域（位置1-20），另一个对应于包含前两个半胱氨酸残基的片段（残基126-143）。用与牛血清白蛋白缀合的肽免疫后，在兔子体内引发特异性抗肽抗体。由此获得的抗肽抗体与受体交叉反应并特异性结合其α亚基。抗肽抗体用于测试肽序列是否对应于α-银环蛇毒素（α-BTX）结合位点。金黄色葡萄球菌 V8 蛋白酶消化分离的受体 α 亚基产生了几个片段。抗肽(1-20)和抗肽(126-143)均结合26-kDa片段，而仅抗肽(126-143)结合17-kDa片段。没有发现这些片段与 alpha-BTX 结合。另一方面，α-BTX 与 18 kDa 片段结合，该片段不与任何一种抗肽抗体发生反应。此外，还发现26-kDa和17-kDa片段含有内切糖苷酶H-敏感寡糖链。我们的结果表明，毒素结合位点位于残基 126-143 之后第一个可能的 V8 蛋白酶切割位点之外：即 Asp-152。该位置与半胱氨酸残基192和/或193非常接近或邻近配体结合位点的可能性一致。
Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site.