Summary
Black lipid membranes were formed with oxidized cholesterol in the presence of either the acetylcholine receptor, purified from the electric organ of the electric rayTorpedo californica or its tryptic digest. In both cases, conductance of cations increased and was dependent on the concentration of the receptor protein. Conductance of Ca++ was dependent on its concentration, but addition of carbamylcholine gave no reproducible or consistent effects. Only in the case of the tryptic digest of the acetylcholine receptor did carbamylcholine and acetylcholine consistently induce monovalent cation selective conductance (P Na, K∶P Cl=4.4). The induced monovalent cationic conductance due to carbamylcholine (10 μm) varied from 10- to over 100-fold. Curare (10 μm) prevented the action of carbamylcholine.
Na-dodecyl sulfate gel electrophoresis of the acetylcholine receptor, before and after tryptic digestion, indicated that this mild enzyme treatment hydrolyzed the receptor molecule subunits. Nevertheless, the receptor molecule retained its full binding of [acetyl-3H]acetylcholine; and analytical gel electrophoresis indicated that it remained intact possibly through hydrogen, hydrophobic and disulfide bonding.
Similar content being viewed by others
References
Carroll, R.C., Eldefrawi, M.E., Edelstein, S.J. 1973. Studies on the structure of the acetylcholine receptor fromTorpedo marmorata.Biochem. Biophys. Res. Commun. 55:864
Chang, H.W. 1974. Purification and characterization of acetylcholine receptor — I fromElectrophorus electricus.Proc. Nat. Acad. Sci. USA 71:2113
Chang, K.J., Triggle, D.J. 1973. Quantitative aspects of drug-receptor interactions. I. Ca2+ and cholinergic receptor activation in smooth muscle: A basic model for drug-receptor interactions.J. Theoret. Biol. 40:125
Davis, B.J. 1963. Disc electrophoresis. II. Methods and application to human serum proteins.Ann. N.Y. Acad. Sci. 121:404
Del Castillo, J., Rodriguez, A., Romero, C.A. 1967. Pharmacological studies on an artificial transmitter-receptor system.Ann. N.Y. Acad. Sci. 144:803
Edelstein, S.J., Beyer, W.B., Eldefrawi, A.T., Eldefrawi, M.E. 1975. Molecular weight of the acetylcholine receptors of electric organs and the effect of Triton X-100.J. Biol. Chem. 250:6101
Eldefrawi, M.E., Britten, A.G., Eldefrawi, A.T. 1971. Acetylcholine binding toTorpedo electroplax: Relationship to acetylcholine receptors.Science 173:338
Eldefrawi, M.E., Britten, A.G., O'Brien, R.D. 1971. Action of organosphosphates on binding of cholinergic ligands.Pestic. Biochem. Physiol. 1:101
Eldefrawi, M.E., Eldefrawi, A.T. 1973a. Purification and molecular properties of the acetylcholine receptor fromTorpedo electroplax.Arch. Biochem. Biophys. 159:362
Eldefrawi, M.E., Eldefrawi, A.T. 1973b. Cooperativities in the binding of acetylcholine to its receptor.Biochem. Pharmacol. 22:3145
Eldefrawi, M.E., Eldefrawi, A.T. 1975. Structure and function of the acetylcholine receptor.Croatica Chemica Acta. (in press)
Eldefrawi, M.E., Eldefrawi, A.T., O'Brien, R.D. 1971. Binding of five cholinergic ligands to housefly brain andTorpedo electroplax: Relationship to acetylcholine receptors.Mol. Pharmacol. 7:104
Eldefrawi, M.E., Eldefrawi, A.T., Penfield, L.A., O'Brien, R.D., Van Campen, D. 1975. Binding of calcium and zinc to the acetylcholine receptor purified fromTorpedo californica.Life Sci. 16:925
Eldefrawi, M.E., Eldefrawi, A.T., Wilson, D.B. 1975. Tryptophan and cysteine residues of the acetylcholine receptors ofTorpedo species. Relationship to binding of cholinergic ligands.Biochemistry 14:4304
Goodall, M.C., Bradley, R.J., Saccomani, G., Romine, W.O. 1974. Quantum conductance changes in lipid bilayer membranes associated with incorporation of acetylcholine receptors.Nature 250:68
Hazelbauer, G.L., Changeux, J.-P. 1974. Reconstitution of a chemically excitable membrane.Proc. Nat. Acad. Sci. USA 71:1479
Jain, M.K. 1974. Studies on a reconstituted acetylcholine receptor system: Effect of agonists.Arch. Biochem. Biophys. 164:20
Karlin, A., Cowburn, D. 1973. The affinity-labeling of partially purified acetylcholine receptor from electric tissue ofElectrophorus.Proc. Nat. Acad. Sci. USA 70:3636
Karlsson, E., Heilbronn, E., Widlund, L. 1972. Isolation of the nicotinic acetylcholine receptor by biospecific chromatography on insolubilizedNaja naja neurotoxin.FEBS Letters 28:107
Kemp, G., Dolly, J.P., Barnard, E.A., Wenner, C.E. 1973. Reconstitution of a partially purified endplate acetylcholine receptor preparation in lipid bilayer membranes.Biochem. Biophys. Res. Commun. 54:607
Klett, R.P., Fulpius, B.W., Cooper, D., Smith, M., Reich, E., Possani, L. 1973. The acetylcholine receptor. I. Purification and characterization of a macromolecule isolated fromElectrophorus electricus.J. Biol. Chem. 248:6841
Leuzinger, W., Schneider, M. 1972. Acetylcholine-induced excitation on bilayers.Experientia 28:256
Lindstrom, J.M., Patrick, J. 1974. Purification of the acetylcholine receptor by affinity chromatography.In: Synaptic Transmission and Neuronal Interactions. M.V.L. Bennett, editor. p. 191. Raven Press, New York
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 192:265
McPhie, P. 1971. Dialysis.Methods Enzymol. 22:23
Meunier, J.-C., Sealock, R., Olsen, R., Changeux, J.-P. 1974. Purification and properties of the cholinergic receptor protein fromElectrophorus electricus electric tissue.Europ. J. Biochem. 45:371
Michaelson, D.M., Raftery, M.A. 1974. Purified acetylcholine receptor: Its reconstitution to a chemically excitable membrane.Proc. Nat. Acad. Sci. USA 71:4768
Michaelson, D., Vandlen, R., Bode, J., Moody, T., Schmidt, J., Raftery, M.A. 1974. Some molecular properties of an isolated acetylcholine receptor ion-translocation protein.Arch. Biochem. Biophys. 156:796
Moore, W.M., Holladay, L.A., Puett, D., Brady, R.N. 1974. On the conformation of the acetylcholine receptor protein fromTorpedo nobiliana.FEBS Letters 45:145
Neumann, E., Nachmansohn, D., Katchalsky, A. 1973. An attempt at an integral interpretation of nerve excitability.Proc. Nat. Acad. Sci. USA 70:727
Parisi, M., Reader, T.A., De Robertis, E. 1972. Conductance properties of artificial lipidic membranes containing a proteolipin fromElectrophorus. Response to cholinergic agents.J. Gen. Physiol. 60:454
Prestipino, G., Ceccarelli, D., Conti, F., Carafoli, E. 1974. Interactions of a mitochondrial Ca2+-binding glycoprotein with lipid bilayer membranes.FEBS Letters 45:99
Romine, W.O., Goodall, M.C., Peterson, J., Bradley, R.J. 1974. The acetylcholine receptor. Isolation of a brain nicotinic receptor and its preliminary characterization in lipid bilayer membranes.Biochim. Biophys. Acta 316:316
Ruiz-Manresa, F., Grundfest, H. 1971. Synaptic electrogenesis in eel electroplaques.J. Gen. Physiol. 57:71
Sanders, D.B., Schleifer, L.S., Eldefrawi, M.E., Norcross, N.L., Cobb, E.B. 1975. An immunologically-induced defect of neuromuscular transmission in rats and rabbits.Ann. N.Y. Acad. Sci. (in press)
Schmidt, J., Raftery, M.A. 1973. Purification of acetylcholine receptors fromTorpedo californica electroplax by affinity chromatography.Biochemistry 12:852
Shamoo, A.E. 1974. Isolation of a sodium-dependent ionophore from (Na++K+)-ATPase preparations.Ann. N.Y. Acad. Sci. 342:389
Shamoo, A.E., Albers, R.W. 1973. Na+-selective ionophoric material derived from electric organ and kidney membranes.Proc. Nat. Acad. Sci. USA 70:1191
Shamoo, A.E., MacLennan, D.H. 1974. A Ca++-dependent and-selective ionophore as part of the Ca+++Mg++-dependent adenosinetriphosphatase of sarcoplasmic reticulum.Proc. Nat. Acad. Sci. USA 71:3522
Shamoo, A.E., Myers, M.M. 1974. Na+-dependent ionophore as part of the small polypeptide of the (Na++K+)-ATPase from eel electroplax membrane.J. Membrane Biol. 19:163
Tien, H.T., Carbone, S., Davidowicz, E.A. 1966. Formation of “black” lipid membranes by oxidation products of cholesterol.Nature 212:719
Weber, K., Osborn, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis.J. Biol. Chem. 244:4406
Zerahn, K. 1971. Active transport of the alkali metals by the isolated mid-gut ofHyalophora cecropia.Phil. Trans. Roy. Soc. London B 262:315
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Shamoo, A.E., Eldefrawi, M.E. Carbamylcholine and acetylcholine-sensitive, cation-selective ionophore as part of the purified acetylcholine receptor. J. Membrain Biol. 25, 47–63 (1975). https://doi.org/10.1007/BF01868567
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01868567