Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots.

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Abstract
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 125-I-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. The association of toxin with the .alpha.-subunit could be inhibited by d-tubocurarine (IC50 [median inhibitory concentration] = 0.9 mM). Removal of high-mannose oligosaccharide chains from the .alpha.-subunit by treatment with endoglycosidase H did not interfere with the observed toxin binding. Isolated, immobilized .alpha.-subunit of the Torpedo acetylcholine receptor can evidently bind .alpha.-bungarotoxin. 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.