Localization of histidine residues relevant for the binding of alpha-bungarotoxin to the acetylcholine receptor alpha-subunit in V8-proteolytic fragments.
Histidine residues have been shown to be critical for alpha-BgTx binding to the acetylcholine receptor (Lacorazza et al., 1992; Bouzat et al., 1993; Lacorazza et al., 1995). Receptor subunits from Discopyge tschudii were modified with diethylpyrocarbonate (DEP). DEP treatment produces a concentration-dependent decrease of [125I] alpha-BgTx binding to the alpha-subunit. The neurotoxin binding capacity was fully restored by adding the nucleophile hydroxylamine. By proteolytic mapping of the alpha-subunit with V8-protease, we determined that the binding capacity to the fragment alpha V8-19 decreased 80% by DEP treatment. In addition, the [125I] alpha-BgTx binding to the same fragment decreased by 70% when the subunits were reduced and affinity-alkylated. We report the N-terminal sequence of both subunits and V8-fragments (alpha V8-10, alpha V8-13, and alpha V8-18), which constitute a first contribution to the knowledge of the primary structure of the Discopyge tschudii receptor. We propose that the fragment alpha V8-19 contains one or more of the histidine residues involved in the alpha-BgTx binding and probably includes the Cys alpha 192-193 disulfide bond. Only two histidine residues are present in the extracellular sequence of Torpedo californica for such fragments: His alpha 186 and alpha 204.
Lacorazza
H D
HD
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET) Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
López
R A
RA
Venera
G D
GD
Biscoglio De Jiménez Bonino
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry*; D004555:Electric Fish; D006639:Histidine / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011950:Receptors, Cholinergic / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D002038:Bungarotoxins; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D006639:Histidine