Summary
The reversible binding of14C-decamethonium (Deca) to excitable microsacs prepared from the electric tissue ofElectrophorus electricus is followed by an ultracentrifugal assay. α-Bungarotoxin, a snake venom toxin, blocks irreversibly the binding of14C-Deca. The displacement is partial. The fraction of14C-Deca displaced by α-bungarotoxin corresponds to molecules of Deca bound to the cholinergic receptor site, whereas the fraction of14C-Deca bound in the presence of α-bungarotoxin corresponds to molecules bound to the catalytic site of acetylcholinesterase (AcChE). The total number of cholinergic receptor sites is found to be close but not identical to the total number of catalytic sites of AcChE.
On the same preparation of microsacs, the binding of14C-Deca and the permeability response corresponding to a given concentration of Deca are measured as a function of increased concentration of Deca. The dose-response curve and the binding curve superimpose almost exactly; in other words, the “apparent” affinity of Deca coincides with its “real” affinity. Displacement of14C-Deca byd-tubocurarine gives an “apparent” affinity ford-tubocurarine which coincides as well with its “real” affinity.
The transport properties of the ionophore controlled by one Deca binding site are estimated.
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Buc, M. H., Buc, H. 1968. Allosteric interactions between AMP and orthophosphate sites on phosphorylase b from rabbit muscle.In: Regulation of Enzyme Activity and Allosteric Interactions. E. Kvamme and A. Pihl, editors. p. 109. Universitetsforlaget, Oslo.
Changeux, J.-P. 1961. The feed-back control mechanism of biosyntheticl-threonine deaminase byl-isoleucine.Cold Spring Harbor Symp. Quant. Biol. 26:313.
— 1966. Responses of acetylcholinesterase fromTorpedo marmorata to salts and curarizing drugs.Mol. Pharmacol. 2:369.
— 1969. Remarks on the symmetry and cooperative properties of biological membranes.In: Symmetry and Function of Biological Systems at the Macromolecular Level. A. Engström and B. Strandberg, editors,Nobel Symp., vol. 11, p. 235. Wiley, Interscience Division, New York.
— Blumenthal, R., Kasai, M., Podleski, T. R. 1970. Conformational transitions in the course of membrane excitation.In: Ciba Symposium, Molecular properties of Drug Receptors. R. Porter and M. O'Connor, editors p. 197. Churchill, London.
—, Kasai, M., Huchet, M., Meunier, J. C. 1970a. Extraction à partir du tissu électrique de gymnote d'une protéine présentant plusieurs propriétés caractéristiques du récepteur physiologique de l'acétylcholine.Compt. Rend. Acad. Sci. Paris 270:2864.
——, Lee, C. Y. 1970b. The use of a snake venom toxin to characterize the cholinergic receptor protein.Proc. Nat. Acad. Sci. 67:1241.
—, Leuzinger, W., Huchet M. 1968. Specific binding of acetylcholine to acetylcholinesterase in the presence of eserine.F.E.B.S. Letters 2:77.
—, Podleski, T. R. 1968. On the excitability and cooperativity of the electroplax membrane.Proc. Nat. Acad. Sci. 59:944.
——, Kasai, M., Blumenthal, R. 1970. Some molecular aspects of membrane excitation studied with the eel electroplax.In: Excitatory Synaptic Mechanisms. P. Andersen and J. Jansen, editors. p. 123. Universitetsforlaget, Oslo.
Changeux, J.-P., Podleski, T. R., Meunier, J. C. 1969. On some structural analogies between acetylcholinesterase and the macromolecular receptor of acetylcholine.J. Gen. Physiol. 54: 225 S.
—, Rubin, M. M. 1968. Allosteric interactions in aspartate transcarbamylase. III. Interpretations of experimental data in terms of the model of Monod, Wyman and Changeux?Biochemistry 7:553.
—, Thiéry, J., Tung, Y., Kittel, C., 1967. On the cooperativity of biological membranes.Proc. Nat. Acad. Sci. 57:335.
Ehrenstein, G., Lecar, H., Nossal, R. 1970. The nature of the negative resistance in bimolecular lipid membranes containing excitability inducing material.J. Gen. Physiol. 55:119.
Gerhart, J. C., Pardee, A. B. 1962. The enzymology of control by feed-back inhibition.J. Biol. Chem. 237:891.
—, Schachman, H. K. 1965. Distinct subunits for the regulation and catalytic activity of aspartate transcarbamylase.Biochemistry 4:1054.
Hille, B. 1970. Ionic channels in nerve membranes.Prog. Biophys. Mol. Biol. 21:1.
Johnson, S. M., Bangham, A. D. 1969. Potassium permeability of single compartment liposomes with and without valinomycin.Biochim. Biophys. Acta 193:82.
Karlin, A. 1967. On the application of a plausible model of allosteric proteins to the receptor for acetylcholine.J. Theoret. Biol. 16:306.
Karlin, A. 1969. Chemical modification of the activity of the acetylcholine receptor.J. Gen. Physiol. 54: 245S.
Karlin, A., Prives, J., Deal, W., Winnik, M. 1970. Counting acetylcholine receptors in the electroplax.In: Ciba Symposium, Molecular Properties of Drug Receptors. R. Porter and M. O'Connor, editors. p. 247. J. and A. Churchill, London.
Kasai, M., Changeux, J.-P., Monnerie, L. 1969.In vitro interaction of 1-anilino-8-naphthalene sulfonate with excitable membranes isolated from the electric organ ofElectrophorus electricus.Biochem. Biophys. Res. Commun. 36:420.
—, Podleski, T. R., Changeux, J.-P. 1970. Some structural properties of excitable membranes labelled by fluorescent probes.F. E. B. S. Letters 13:7.
Kennedy, E. 1969. Studies on the lactose transport system inEscherichia coli. J. Gen. Physiol. 54:91 S.
Kremzner, L., Wilson, I. B. 1964. A partial characterization of acetylcholinesterase.Biochemistry 3:1902.
Lee, C. Y., Chang, C. C. 1966. Modes of action of purified toxins from Elapid venoms on neuromuscular transmission.Mem. Inst. Butantan. Simp. Internac. 33:555.
Leuzinger, W., Baker, A. 1967. Acetylcholinesterase. I. Large scale purification, homogeneity, amino acid analysis.Proc. Nat. Acad. Sci. 57:446.
——, Cauvin, E. 1968. Acetylcholinesterase. II. Crystallization, absorption spectra, isoionic point.Proc. Nat. Acad. Sci. 59:620.
—, Goldberg, M., Cauvin, E. 1969. Molecular properties of acetylcholinesterase.J. Mol. Biol. 40:217.
Massoulié, J., Riéger, F. 1969. L'acétylcholinestérase des organes électriques de poissons (torpille et gymnote); complexes membranaires.Europ. J. Biochem. 11:441.
Meunier, J.-C., Huchet, M., Boquet, P., Changeux, J.-P. 1971. Séparation de la protéine réceptrice de l'acétylcholine et de l'acétylcholinestérase.Compt. Rend. Acad. Sci. (Paris) 272:117 D.
Monod, J., Changeux, J.-P., Jacob, J. 1963. Allosteric proteins and cellular control systems.J. Mol. Biol. 6:306.
—, Wyman, J., Changeux, J.-P. 1965. On the nature of allosteric transitions: A plausible model.J. Mol. Biol. 12:88.
Moore, J. W., Narahashi, T., Shaw, T. I. 1967. An upper limit to the number of sodium channels in nerve membrane.J. Physiol. 188:99.
Mueller, P., Rudin, D. O. 1968. Resting and action potentials in experimental bimolecular lipid membranes.J. Theoret. Biol. 18:222.
—— 1968. Action potentials induced in bimolecular lipid membranes.Nature 217:713.
Nachmansohn, D. 1959. Chemical and Molecular Basis of Nerve Activity. Academic Press, New York.
Nachmansohn, D. 1969. Proteins of excitable membranes.J. Gen. Physiol. 54: 187 S.
O'Brien, R. D., Gilmour, L. P. 1969. A muscarone-binding material in electroplax and its relation to the acetylcholine receptor. I. Centrifugal assay.Proc.Nat. Acad. Sci. 63:496.
——, Eldefrawi, M. E. 1970. A muscarone-binding material in electroplax and its relation to the acetylcholine receptor. II. Dialysis assay.Proc. Nat. Acad. Sci. 65:438.
Post, R., Sen, A. K., Rosenthal, A. S. 1965. A phosphorylated intermediate in adenosine triphosphate dependent sodium and potassium transport across kidney membranes.J. Mol. Chem. 240:1437.
Wahl, P., Kasai, M., Changeux, J.-P. 1971. A study on the motion of proteins in excitable membrane fragments by a nanosecond fluorescence polarization spectroscopy.Europ. J. Biochem. 18:332.
Whitehead, E. 1970. The regulation of enzyme activity and allosteric transition.Prog. Biophys. Mol. Biol. 21:321.
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Kasai, M., Changeux, JP. In vitro excitation of purified membrane fragments by cholinergic agonists. J. Membrain Biol. 6, 58–80 (1971). https://doi.org/10.1007/BF01874114
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DOI: https://doi.org/10.1007/BF01874114