title: Partial characterization of solubilized platelet imipramine binding sites using a new probe, [3H]3-cyanoimipramine ([3H]Ro 11-2465).
abstract: Evidence suggests that [3H]imipramine labels the recognition site of the neuronal 5-hydroxytryptamine uptake mechanism. We are investigating the linkage between these binding sites and the carrier by biochemical characterization. [3H]Imipramine-labelled sites have been solubilized from outdated human platelets using the detergent digitonin. [3H]3-Cyanoimipramine binds persistently to these sites in the presence of Na+ at 4 degrees C. At higher temperatures, and in the absence of Na+, this ligand acts reversibly. We report the use of this pseudo-irreversible ligand in the initial molecular characterization of the recognition molecule. To confirm that this ligand occupies the [3H]imipramine-labelled sites, human platelets were prelabelled with 3-cyanoimipramine before incubating with [3H]imipramine. Only low affinity [3H]imipramine binding remained. The majority of the 3-cyanoimipramine was irreversibly bound under these conditions as shown by the use of the 3H compound. Gel permeation chromatography of [3H]3-cyanoimipramine-prelabelled platelet membranes solubilized with digitonin indicated a Stokes' radius of 6.3 nm. This is larger than values previously determined for cholate-solubilized sites. We conclude that [3H]3-cyanoimipramine will be useful for further purification and reconstitution studies.
authors.forename: A
authors.initials: A
authors.lastname: Davis
authors.forename: J M
authors.initials: JM
authors.lastname: Morris
authors.forename: S W
authors.initials: SW
authors.lastname: Tang
mesh_terms: D001667:Binding, Competitive / Q000187:drug effects; D001792:Blood Platelets / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002352:Carrier Proteins; D002850:Chromatography, Gel; D006801:Humans; D007099:Imipramine / Q000031:analogs & derivatives* / Q000097:blood*; D066298:In Vitro Techniques; D007700:Kinetics; D011955:Receptors, Drug; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012702:Serotonin Antagonists; D013997:Time Factors
chemical_list: D001798:Blood Proteins; D002352:Carrier Proteins; D011955:Receptors, Drug; D017981:Receptors, Neurotransmitter; D012702:Serotonin Antagonists; C027775:imipramine receptor; C024316:cianopramine; D007099:Imipramine