J. Biol. Chem., Vol. 258, Issue 17, 10263-10271, Sep, 1983
Interaction of cholinergic ligands with the purified acetylcholine receptor protein. I. Equilibrium binding studies
H Prinz and A Maelicke
We have studied the binding equilibria of two fluorescent ligands and
several nonfluorescent cholinergic ligands with the purified acetylcholine
receptor from Electrophorus electricus. The assay was based on the
specifically cholinergic and reversible quenching of fluorescence observed
upon complex formation between the receptor protein and
N-7-(4-nitrobenzo-2-oxa-1,3-diazole)-5-aminopentanoic acid
beta-(N-trimethylammonium) ethyl ester. This way, a large body of accurate,
true equilibrium data was obtained. We find 1) all ligands studied compete
for the same number of binding sites at the receptor; 2) agonists compete
for half of these sites with high affinity and for the other half of these
sites with significantly lower affinity; 3) antagonists compete for all of
these sites with one affinity; and 4) in the presence of disulfide reducing
agents, the binding patterns of some agonists and antagonists are changed
in accordance with the electrophysiological changes observed under the same
conditions with Rana pipiens Sartorius muscle fibers. Our studies indicate
that the mechanism of ligand recognition is still functional at equilibrium
and is not subject to the presence of an intact membrane environment.
Furthermore, the existence of two types of agonist sites at every receptor
molecule excludes most of the presently discussed two-state models as the
basis for a mechanism of receptor-ligand interaction. To explain sigmoidal
dose-response curves, a two-site model is already sufficient.
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