Molecular Pharmacology, Vol 7, 554-566, Copyright © 1971 by the American Society for Pharmacology and Experimental Therapeutics

Regulation of Membrane-Bound Acetylcholinesterase Activity by Bisquaternary Nitrogen Compounds

¹ Institut für Biochemie, Universität Mainz, Germany

The inhibitory effect of bisquaternary compounds on erythrocyte membrane-bound acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) was investigated by kinetic methods. The membranes were obtained by osmotic hemolysis of human erythrocytes. A special photometric method was used for measurements, which made it possible to record the enzyme reaction in the steady-state phase. Incontrast to former investigations, in which the reaction medium contained Na⁺ and Mg²⁺ ions at rather high concentrations, these ions were omitted in the present experiments.

Kinetic analysis of the results yielded the following types of inhibitory mechanisms: d-tubocurarine and gallamine, partially competitive—partially noncompetitive; succinylbischoline, partially competitive-noncompetitive ("mixed inhibition"). The noncompetitive portion of the mechanism of all compounds investigated is interpreted in the following way. d-Tubocurarine, gallamine, and succinylbischoline are bound to a site distinct from the active site of acetylcholinesterase. This site, called the "regulatory site," does not show catalytic activity and contains at least two negative charges. The presence of noncompetitive inhibition suggests that the binding of these effectors to the regulatory site must be combined with a conformational change, which affects the enzyme reaction rate negatively in the case of d-tubocurarine, gallamine, and succinylbischoline. The phenomenon of substrate concentration-dependent activation and inhibition, which is observed with the effector pentamethonium, is explicable only if the existence of a ternary enzyme-substrate-inhibitor (ESI) complex is accepted. This means that the reaction mechanism is expected to be partially noncompetitive in this case too, and consequently pentamethonium has to be designated as an allosteric effector. The regulatory site, whose existence is proved by the presence of the noncompetitive part of the reaction mechanism, could be identical with the cholinergic receptor defined by pharmacological experiments.