Lipid-dependent recovery of alpha-bungarotoxin and monoclonal antibody binding to the purified alpha-subunit from Torpedo marmorata acetylcholine receptor. Enhancement by noncompetitive channel blockers.
Recently the purified alpha-subunit from Torpedo marmorata acetylcholine receptor was shown to bind alpha-bungarotoxin with a KD approximately 3 nM in the presence of sodium dodecyl sulfate (Tzartos, S.J., and Changeux, J.P. (1983) EMBO J. 2, 381-387). Here we describe a further significant step toward renaturation of the alpha-subunit as judged by toxin and monoclonal antibody binding. Purified T. marmorata receptor subunits were diluted with 1% lipids (asolectin) plus 0.5% Na+ cholate. An anion-exchange resin eliminated most of the detergents, leaving approximately 0.1% Na+ cholate and the lipids. After this treatment, about 20% of the alpha-subunit recovered (but not the beta-, gamma-, or delta-subunit) exhibited a high affinity for radioiodinated alpha-bungarotoxin with a KD approximately 0.5 nM. The 34,000- and 27,000-dalton proteolytic peptides of the alpha-subunit conserved this lipid-dependent toxin binding. Unlabeled alpha-toxins, hexamethonium, and carbamylcholine competed with alpha-bungarotoxin for the renatured alpha-subunit. Noncompetitive channel blockers doubled the lipid-dependent toxin-binding capacity of the alpha-subunit but had no effect on the 27,000-dalton peptide. The binding of several monoclonal antibodies to the main immunogenic region (which is particularly sensitive to denaturation) significantly increased. In particular, binding of antibody 16 changed from 1% to denatured to 100% to the lipid-renaturated alpha-subunit. The binding of these antibodies was lost with the lipid-renatured 34,000- and 27,000-dalton peptides.
D000818:Animals; D000911:Antibodies, Monoclonal; D002038:Bungarotoxins / Q000378:metabolism*; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D003650:Decamethonium Compounds / Q000494:pharmacology; D007473:Ion Channels / Q000187:drug effects*; D050356:Lipid Metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D014101:Torpedo
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D002793:Cholic Acids; D003650:Decamethonium Compounds; D007473:Ion Channels; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D012967:Sodium Dodecyl Sulfate; D019826:Cholic Acid