Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor.
We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.
Department of Physiology, University of California, San Francisco 94143-0444.
A J
AJ
Dowding
Z W
ZW
Hall
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism; D011863:Radioimmunoassay; D011950:Receptors, Cholinergic / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002038:Bungarotoxins; D011950:Receptors, Cholinergic