Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.
Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four 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-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, 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-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor.
D000818:Animals; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D010450:Endopeptidases; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo
D002038:Bungarotoxins; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D010450:Endopeptidases