Two-step isomerization of quinuclidinyl benzilate-muscarinic receptor complex.

ABSTRACT

A kinetic analysis was made of the dissociation reaction of the muscarinic receptor-l-[(3)H]quinuclidinyl benzilate complex at 25 degrees C in 0.05 M K-phosphate buffer. The course of the reaction was followed by the decrease in the concentration of the membrane-bound radiolabelled antagonist while rebinding was prevented by the excess of nonradioactive quinuclidinyl benzilate. It was found that both bi- and mono-exponential kinetic curves of the process can be observed, depending on the time moment when the dissociation reaction is started. If the receptor-ligand complex had been incubated for a sufficiently long time before the excess of the nonradioactive ligand was added to "displace" the radioactive ligand from the complex, the dissociation reaction followed the first-order kinetics. The bi-exponential kinetics of the dissociation process was obtained if the displacement was started within a short time interval after the complex formation between the receptor and l-[(3)H]quinuclidinyl benzilate. The data obtained were analysed within the framework of a reaction scheme containing two consecutive isomerization steps of the receptor-antagonist complex. The "isomerized" receptor-ligand complexes differ in their dissociation rate and therefore their interconversion changes the observed kinetic behaviour of the dissociation reaction of the receptor-ligand complex.