High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate.
Intact nicotinic acetylcholine receptor (AChR) tightly binds alpha-bungarotoxin. The two toxin-binding sites are presumed to be on the two alpha-subunits, either on or near the ACh-binding sites. Isolated alpha-subunits have been found to maintain weak binding to alpha-bungarotoxin (KD approximately 0.2 microM). We describe here conditions under which the alpha-subunit and a 27,000-dalton proteolytic peptide bound alpha-bungarotoxin with high affinity. The four subunits of Torpedo marmorata AChR, as well as several proteolytic peptides of the alpha-subunit, were first purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. We found that the purified alpha-subunit (but not the beta-, gamma- or delta-subunits) and its 27,000-dalton peptide specifically bound 125I-labeled alpha-bungarotoxin with KD approximately 3 and 6 nM, i.e., about two orders of magnitude lower than the intact AChR. Nearly 100% of the sites were recovered. The recovery of this high affinity binding required the presence of SDS (approximately 0.02%) but non-denaturing detergents had a strongly inhibitory effect. Unlabeled alpha-toxins competed with labeled alpha-bungarotoxin, alpha-bungarotoxin being more effective than all the other toxins tested. Decamethonium and hexamethonium competed efficiently with alpha-bungarotoxin binding but carbamylcholine had only a weak effect. The main immunogenic region of the AChR was only partially preserved since conformation-dependent monoclonal antibodies to this region bound the alpha subunit-toxin complexes, but much less efficiently than the intact AChR. We conclude that SDS can be advantageous to the recovery of high toxin binding to the alpha subunit which still has not completely recovered its native conformation.
Tzartos
S J
SJ
Unité de Neurobiologie Moléculaire, Institut Pasteur, Paris, France.
Changeux
J P
JP
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002038:Bungarotoxins / Q000378:metabolism*; D003902:Detergents; D007457:Iodine Radioisotopes; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D014101:Torpedo / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D003902:Detergents; D007457:Iodine Radioisotopes; D010455:Peptides; D011978:Receptors, Nicotinic; D012967:Sodium Dodecyl Sulfate