title: Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.
abstract: 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.
authors.forename: D
authors.initials: D
authors.lastname: Neumann
authors.forename: J M
authors.initials: JM
authors.lastname: Gershoni
authors.forename: M
authors.initials: M
authors.lastname: Fridkin
authors.forename: S
authors.initials: S
authors.lastname: Fuchs
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins; D000939:Epitopes; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000276:immunology*
chemical_list: D002038:Bungarotoxins; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D000109:Acetylcholine