Preservation of Functional Properties of the Acetylcholine Receptor After Solubilization
Langley’s receptive substance and the prototype for all other neurotransmitter and hormone receptors is the acetylcholine receptor, an integral membrane protein that remains tightly associated with its surrounding membrane during cell fractionation procedures. After solubilization with non-ionic detergents the receptor protein can be purified to apparent homogeneity, thus providing a water soluble but micro-heterogenous preparation for biochemical studies. As a “cell-free” system the purified receptor protein lacks its natural environment and may therefore function differently than in the membrane bound state. A comparison of results from earlier equilibrium binding studies and physiological measurements supports this view (for a review see Maelicke et al. 1977; Heidmann and Changeux 1978): For agonists the shape of binding curves did not resemble their sigmoidal dose-response curves and their equilibrium dissociation constants were generally much lower than the concentrations for half-maximal response. With the fluorescent acetylcholine analogs, NBD-n-acylcholines, recently synthesized in our laboratory (Jürss et al. 1979 a, b) we were able to obtain more accurate binding data for representative cholinergic ligands; they appear to be consistent with the physiology of the cholinergic system: (1) Agonists have two affinities of binding to the solubilized receptor. One of these is the high affinity previously observed, the other one is a low affinity in good agreement with the concentration for half-maximal response of the particular agonist. (2) Antagonists bind to the same number of sites with one affinity only. (3) Agonists bind by a sequential (anti-cooperative) mechanism to the solubilized receptor. With the assumption of the second (low affinity) sites initiating response, the discrepancies between KD-values and concentrations of half-maximal response disappear and sigmoidal dose-response curves can be explained (Prinz and Maelicke 1980 b).
KeywordsAcetylcholine Receptor Second Order Reaction Agonist Binding Equilibrium Dissociation Constant High Affinity Site
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