Determination of the primary amino acid sequence specifying the alpha-bungarotoxin binding site on the alpha subunit of the acetylcholine receptor from Torpedo californica.
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.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D003208:Concanavalin A / Q000378:metabolism; D006596:Hexosaminidases; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic / Q000032:analysis* / Q000378:metabolism; D014101:Torpedo
D002038:Bungarotoxins; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D003208:Concanavalin A; D006596:Hexosaminidases