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 (KD, 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.
D000818:Animals; D002038:Bungarotoxins / Q000302:isolation & purification / Q000378:metabolism*; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D008024:Ligands; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000302:isolation & purification / Q000378:metabolism; D014101:Torpedo
D002038:Bungarotoxins; D005782:Gels; D008024:Ligands; D010455:Peptides; D011950:Receptors, Cholinergic; D011978:Receptors, Nicotinic; D003101:Collodion