Purification and characterization of a nicotinic acetylcholine receptor from chick brain.
Immunohistochemical studies have previously shown that both the chick brain and chick ciliary ganglion neurons contain a component which shares antigenic determinants with the main immunogenic region of the nicotinic acetylcholine receptor from electric organ and skeletal muscle. Here we describe the purification and initial characterization of this putative neuronal acetylcholine receptor. The component was purified by monoclonal antibody affinity chromatography. The solubilized component sediments on sucrose gradients as a species slightly larger than Torpedo acetylcholine receptor monomers. It was affinity labeled with bromo[3H]acetylcholine. Labeling was prevented by carbachol, but not by alpha-bungarotoxin. Two subunits could be detected in the affinity-purified component, apparent molecular weights 48 000 and 59 000. The 48 000 molecular weight subunit was bound both by a monoclonal antibody directed against the main immunogenic region of electric organ and skeletal muscle acetylcholine receptor and by antisera raised against the alpha subunit of Torpedo receptor. Evidence suggests that there are two alpha subunits in the brain component. Antisera from rats immunized with the purified brain component exhibited little or no cross-reactivity with Torpedo electric organ or chick muscle acetylcholine receptor. One antiserum did, however, specifically bind to all four subunits of Torpedo receptor. Experiments to be described elsewhere (J. Stollberg et al., unpublished results) show that antisera to the purified brain component specifically inhibit the electrophysiological function of acetylcholine receptors in chick ciliary ganglion neurons without inhibiting the function of acetylcholine receptors in chick muscle cells. All of these properties suggest that this component is a neuronal nicotinic acetylcholine receptor with limited structural homology to muscle nicotinic acetylcholine receptor.
Whiting
P J
PJ
Lindstrom
J M
JM
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001921:Brain / Q000378:metabolism*; D002038:Bungarotoxins / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002642:Chick Embryo; D002645:Chickens; D002846:Chromatography, Affinity; D004557:Electric Organ / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000196:embryology / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011978:Receptors, Nicotinic / Q000302:isolation & purification* / Q000378:metabolism; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002038:Bungarotoxins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D002217:Carbachol; D000109:Acetylcholine