INTERACTION OF [ALPHA-BUNGAROTOXIN-I-125 WITH ACETYLCHOLINE-RECEPTOR FROM TORPEDO-CALIFORNICA

The preparation and characterization of an iodinated derivative of α-bungarotoxin are described. A general method for investigation of the kinetic homogeneity of a radiolabeled toxin is presented. The kinetics of iodinated α-bungarotoxin binding to acetylcholine receptor from Torpedo californica are quantitatively studied in the presence and absence of cholinergic ligands by a DEAE filter disc assay (Schmidt, J., & Raftery, M. A. (1973) Anal. Biochem. 52, 349) which is described in detail. In the case of membrane-bound receptor, the concentration dependence of the kinetics of toxin binding was determined up to the experimentally attainable limit of 3 μM. The kinetics followed a one-step association mechanism with a single class of (non-interacting) binding sites in the membranes. The association rate constant was (1.2 ± 0.1) × 104 M-1s-1, and the lifetime of the complex was longer than 2 days. Preequilibration of the membrane-bound receptor with cholinergic ligands resulted in an apparently competitive inhibition of toxin binding. This inhibition is adequately described by simple hyperbolas without indication of heterogeneous or interacting ligand binding sites on the membrane-bound receptor. In the case of solubilized, purified receptor, the kinetics with toxin in excess were faster than those for the membrane-bound receptor and were biphasic. Perturbation of the native environment of the receptor may be the origin of the heterogeneous toxin binding kinetics. © 1979, American Chemical Society. All rights reserved.