Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

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Abstract
Proteolytic fragments of the .alpha. subunit of the acetylcholine receptor retain the ability to bind .alpha.-bungarotoxin [.alpha.-BTX] following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The .alpha. subunit of the acetylcholine receptor of T. californica electric organ was digested with 4 proteases: Staphylococcus aureus V-8 protease, papain, bromelain and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled .alpha.-BTX and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The MW of the fragments binding toxin ranged from 7-34 kD[kilodalton], with major groupings in the 8-, 18- and 28-kD ranges. The apparent affinity of the fragments for .alpha.-BTX as determined from the IC50 value was 6.7 .times. 10-8 M. The labeling of fragments with .alpha.-BTX could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-.alpha.-benzyltrimethylammonium iodide. Immobilized proteolytic fragments as small as 1/5 the size of the .alpha. subunit retain the structural characteristics necessary for binding .alpha.-BTX, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the .alpha.-BTX binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.