Analysis of receptor-ligand interactions using nitrocellulose gel transfer: Application to Torpedo acetylcholine receptor and alpha-bungarotoxin

A nitrocellulose-gel transfer technique has been adapted to study the interaction of a polypeptide ligand with individual receptor subunits. The acetylcholine receptor isolated from Torpedo californica has been separated into its subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred in a renaturing environment to nitrocellulose sheets. The sheets were incubated with ¹²⁵I-alpha-bungarotoxin and autoradiographed. A single receptor polypeptide, the alpha subunit (40K) bound the labeled toxin. This binding was demonstrated to be both saturable and specific, although the affinity of ¹²⁵I-alpha-bungarotoxin (K_D, 165 nm) and the potency of d-tubocurarine to displace this binding (IC₅₀, 1 mm) were both reduced by several orders of magnitude when compared to the native receptor.