Temperature-sensitive conformational changes in [3H]imipramine binding sites and the involvement of...

Recently, a reversible, temperature-sensitive conformational change has been observed which alters [3H]imipramine binding parameters. I now report that the conformational change probably occurs within the binding site protein and is not due to allosteric interactions or altered ligand kinetics. Evidence comes from the identification of sulphur-containing bonds in the binding site molecule which are differentially exposed at different incubation temperatures. Binding of [3H]imipramine to outdated human platelets is maximal at 4 degrees C; when the incubation temperature is raised to 23 degrees C, a loss of affinity is observed. N-Ethylmaleimide, an alkylator of sulphydryl groups, could completely inhibit the binding at both temperatures but did so much faster at 23 degrees C than at 4 degrees C. Dithioerythritol (DTE) and beta-mercaptoethanol reduce disulphide bonds and were found to enhance [3H]imipramine binding. Again, these reagents were more potent at 23 degrees C than at 4 degrees C. None of these reagents caused these effects by interacting with the ligand. Simultaneous preincubation with excess fluoxetine could prevent the action of DTE on specific [3H]imipramine binding. It is concluded that sulphur-containing bonds are close to, or located at the [3H]imipramine binding site, and their conformation is sensitive to changing temperatures.