Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

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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 1st corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the 1st 2 cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies 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. The toxin-binding site apparently 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.