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Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.

By P T Wilson, T L Lentz and E Hawrot

Abstract

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

Topics: Research Article
Year: 1985
DOI identifier: 10.1073/pnas.82.24.8790
OAI identifier: oai:pubmedcentral.nih.gov:391523
Provided by: PubMed Central
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