Molecular Pharmacology, Vol 2, 369-392, Copyright © 1966 by the American Society for Pharmacology and Experimental Therapeutics

Responses of Acetylcholinesterase from Torpedo marmorata to Salts and Curarizing Drugs

¹ Service de Biochimie Cellulaire, Institut Pasteur, Paris, France; Department of Biology, New York State University, Buffalo, New York, and Virus Laboratory, University of California, Berkeley, California

The structure and catalytic activity of acetylcholinesterase (Acetylcholine acetyl-hydrolase, EC 3.1.1.7) (AChE) from Torpedo marmorata depend upon the ionic strength, /2, of the environment. The sedimentation coefficient of the enzyme is 14 S at /2 = 0.3, but is polydisperse in the range of 10-80 S at /2 = 0.003. The optimal velocity of the reaction catalyzed by AChE increases with ionic strength, while under the same conditions the affinity for the substrate and for several reversible competitive inhibitors decreases. The relative decrease of affinity as a consequence of increased ionic strength is higher for inhibitor molecules containing two quaternary ammonium ions than for compounds containing a single quaternary ammonium group. Among the monoquaternary inhibitors, this decrease is greater for phenyltrimethylammonium than for its 3-hydroxy analog.

In solutions of low salt concentration (/2 = 0.003) significant affinity of the enzyme for two pachycurares, flaxedil and d-tubocurarine, can be demonstrated. Both compounds produce partial inhibition of AChE activity, antagonize its inhibition by reversible competitive inhibitors including some leptocurares, and enhance the inhibition by 3-hydroxyphenyltrimethylammonium. By measuring the degree of protection of AChE against thermal inactivation, the dissociation constant for the flaxedil-enzyme complex can be estimated to be about 3 x 10^-7 M.

These observations and their possible physiological significance are interpreted in terms of conformational alterations of the AChE molecule in response to the binding of the pharmacologic agents.

Note:
ACKNOWLEDGMENTS The author is greatly indebted to Dr. France Tazieff-Depierre for her contributions to the early stages of this research and gifts of most of the pharmacologic agents and to Mrs. Merry Rubin for her extensive assistance in the preparation of the manuscript. He thanks Drs. P. Ascher, R. Couteaux, J. C. Gerhart, and J. Monod for their comments and suggestions after reading the manuscript. This investigation was supported in part by grants from the National Institutes of Health, National Science Foundation, Jane Coffin Childs Memorial Fund, Délégation Générale à la Recherche Scientifique et Technique, and U.S. Public Health Service research grant GM 12159 from the National Institutes of General Medical Sciences. A portion of the research was carried out during tenure of an Eleanor Roosevelt International Cancer Fellowship administered by the International Union Against Cancer.

This article has been cited by other articles:

				J. Koehnke, X. Jin, E. C. Budreck, S. Posy, P. Scheiffele, B. Honig, and L. Shapiro Crystal structure of the extracellular cholinesterase-like domain from neuroligin-2 PNAS, February 12, 2008; 105(6): 1873 - 1878. [Abstract] [Full Text] [PDF]

				Y. Bourne, Z. Radic, G. Sulzenbacher, E. Kim, P. Taylor, and P. Marchot Substrate and Product Trafficking through the Active Center Gorge of Acetylcholinesterase Analyzed by Crystallography and Equilibrium Binding J. Biol. Chem., September 29, 2006; 281(39): 29256 - 29267. [Abstract] [Full Text] [PDF]

				A. A. Kousba, L. G. Sultatos, T. S. Poet, and C. Timchalk Comparison of Chlorpyrifos-Oxon and Paraoxon Acetylcholinesterase Inhibition Dynamics: Potential Role of a Peripheral Binding Site Toxicol. Sci., August 1, 2004; 80(2): 239 - 248. [Abstract] [Full Text] [PDF]

				Y. Bourne, H. C. Kolb, Z. Radic, K. B. Sharpless, P. Taylor, and P. Marchot Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation PNAS, February 10, 2004; 101(6): 1449 - 1454. [Abstract] [Full Text] [PDF]

				C. B. Millard, V. L. Shnyrov, S. Newstead, I. Shin, E. Roth, I. Silman, and L. Weiner Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones Protein Sci., October 1, 2003; 12(10): 2337 - 2347. [Abstract] [Full Text] [PDF]

				J. Shi, Z. Radic', and P. Taylor Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69-Cys96, of Mouse Acetylcholinesterase J. Biol. Chem., November 1, 2002; 277(45): 43301 - 43308. [Abstract] [Full Text] [PDF]

				Y. Boublik, P. Saint-Aguet, A. Lougarre, M. Arnaud, F. Villatte, S. Estrada-Mondaca, and D. Fournier Acetylcholinesterase engineering for detection of insecticide residues Protein Eng. Des. Sel., January 1, 2002; 15(1): 43 - 50. [Abstract] [Full Text] [PDF]

				S. A. Kardos and L. G. Sultatos Interactions of the Organophosphates Paraoxon and Methyl Paraoxon with Mouse Brain Acetylcholinesterase Toxicol. Sci., November 1, 2000; 58(1): 118 - 126. [Abstract] [Full Text] [PDF]

				V. Marcel, S. Estrada-Mondaca, F. Magne, J. Stojan, A. Klaebe, and D. Fournier Exploration of the Drosophila Acetylcholinesterase Substrate Activation Site Using a Reversible Inhibitor (Triton X-100) and Mutated Enzymes J. Biol. Chem., April 14, 2000; 275(16): 11603 - 11609. [Abstract] [Full Text] [PDF]

				N. Morel, S. Bon, H. M. Greenblatt, D. Van Belle, S. J. Wodak, J. L. Sussman, J. Massoulié, and I. Silman Effect of Mutations within the Peripheral Anionic Site on the Stability of Acetylcholinesterase Mol. Pharmacol., June 1, 1999; 55(6): 982 - 992. [Abstract] [Full Text]

				O. I. Casanueva, T. Garcia-Huidobro, E. O. Campos, R. Aldunate, J. Garrido, and N. C. Inestrosa A Major Portion of Synaptic Basal Lamina Acetylcholinesterase Is Detached by High Salt- and Heparin-containing Buffers from Rat Diaphragm Muscle and Torpedo Electric Organ J. Biol. Chem., February 13, 1998; 273(7): 4258 - 4265. [Abstract] [Full Text] [PDF]

				X. Cousin, S. Bon, N. Duval, J. Massoulie, and C. Bon Cloning and Expression of Acetylcholinesterase from Bungarus fasciatus Venom. A NEW TYPE OF COOH-TERMINAL DOMAIN; INVOLVEMENT OF A POSITIVELY CHARGED RESIDUE IN THE PERIPHERAL SITE J. Biol. Chem., June 21, 1996; 271(25): 15099 - 15108. [Abstract] [Full Text] [PDF]

				A. Ordentlich, D. Barak, C. Kronman, N. Ariel, Y. Segall, B. Velan, and A. Shafferman Contribution of Aromatic Moieties of Tyrosine 133 and of the Anionic Subsite Tryptophan 86 to Catalytic Efficiency and Allosteric Modulation of Acetylcholinesterase J. Biol. Chem., February 3, 1995; 270(5): 2082 - 2091. [Abstract] [Full Text] [PDF]

				J. Sussman, M Harel, F Frolow, C Oefner, A Goldman, L Toker, and I Silman Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein Science, August 23, 1991; 253(5022): 872 - 879. [Abstract] [PDF]

				J. Changeux, A Devillers-Thiery, and P Chemouilli Acetylcholine receptor: an allosteric protein Science, September 21, 1984; 225(4668): 1335 - 1345. [Abstract] [PDF]

				L. Brochier, Y. Pontie, M. Willson, S. Estrada-Mondaca, J. Czaplicki, A. Klaebe, and D. Fournier Involvement of Deacylation in Activation of Substrate Hydrolysis by Drosophila Acetylcholinesterase J. Biol. Chem., May 18, 2001; 276(21): 18296 - 18302. [Abstract] [Full Text] [PDF]

				G. V. De Ferrari, W. D. Mallender, N. C. Inestrosa, and T. L. Rosenberry Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites J. Biol. Chem., June 22, 2001; 276(26): 23282 - 23287. [Abstract] [Full Text] [PDF]