Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.
The direct binding of alpha-bungarotoxin to the alpha subunit of the acetylcholine receptor from Torpedo electric organ immobilized onto protein blots was demonstrated. Protein blots were prepared by electrophoretically transferring resolved acetylcholine receptor subunits from 10% polyacrylamide gels onto Zetabind, positively charged nylon membrane filters. Such blots, when incubated with 125I-labeled alpha-bungarotoxin, washed, and autoradiographed, gave rise to a single labeled band corresponding to the alpha subunit of the receptor. The labeling with alpha-bungarotoxin could be inhibited by pretreating the receptor-containing membranes with the affinity ligand 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. In addition, the association of toxin with the alpha subunit could be inhibited by d-tubocurarine (IC50 = 0.9 mM). Furthermore, removal of high-mannose oligosaccharide chains from the alpha subunit by treatment with endoglycosidase H did not interfere with the observed toxin binding. Thus it is demonstrated that isolated, immobilized alpha subunit of the Torpedo acetylcholine receptor can bind alpha-bungarotoxin. However, the observed binding of alpha-bungarotoxin to immobilized alpha subunit is reduced in affinity to 1/1,000 to 1/10,000 of that obtained with native receptor. The endoglycosidase H-susceptible oligosaccharide side chain(s) is not required for this interaction. Binding of alpha-bungarotoxin is to the physiologically relevant acetylcholine binding site as defined by affinity ligand alkylation.
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo; D014403:Tubocurarine / Q000494:pharmacology
D002038:Bungarotoxins; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D014403:Tubocurarine