Identification of exposed and buried determinants of the membrane-bound acetylcholine receptor from Torpedo californica.
The ability of five rabbit anti-acetylcholine receptor antisera to recognize the membrane-bound receptor from Torpedo californica has been investigated. Two antisera, raised against affinity-purified native receptor, react extensively with purified receptor-rich membrane vesicles. Since the membrane vesicles are impermeable to macromolecules and are oriented right side out, these two antisera recognize predominantly extracellular determinants. Two antisera against sodium dodecyl sulfate denatured receptor and one against purified delta subunit react poorly with the membrane-bound receptor. Only 10-20% of the determinants recognized by these antisera are accessible to antibodies when the receptor is membrane bound. Many of the latent sites can be exposed by permeabilizing the vesicles with saponin, by alkaline extraction of the membranes to remove peripheral proteins, or by a combination of these two treatments. These treatments neither solubilize the receptors nor interfere with their ability to undergo agonist-induced affinity changes. Subunit analysis of the sites on the membrane-bound receptor that are accessible to antibodies indicates that the alpha, beta, and delta chains possess extracellular determinants. Buried sites are present on all four of the subunits. Saponin permeabilization makes latent sites accessible on alpha and delta while alkaline extraction uncovers determinants on alpha, gamma, and delta. Treatment of membranes by both procedures reveals sites on beta, gamma, and delta that are not uncovered by either treatment alone. This study, in conjunction with results from other laboratories demonstrating that the gamma chain is extracellularly exposed, suggests that all four subunits are transmembrane proteins.
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D000936:Antigen-Antibody Complex; D002038:Bungarotoxins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D007106:Immune Sera; D007700:Kinetics; D011950:Receptors, Cholinergic / Q000276:immunology* / Q000378:metabolism; D014101:Torpedo
D000936:Antigen-Antibody Complex; D002038:Bungarotoxins; D000939:Epitopes; D007106:Immune Sera; D011950:Receptors, Cholinergic; D000109:Acetylcholine