Reconstitution of [3H]3-cyanoimipramine ([3H]Ro 11-2465) sites solubilised from human platelets

Abstract

[3H]3-Cyanoimipramine has been characterised as a pseudoirreversible ligand at imipramine binding sites making it useful for molecular characterisation studies. Using this ligand, I now report that it is possible to solubilise the binding site molecule with cholate. Gel permeation chromatography on a Sepharose 4B column equilibrated in 0.1% cholate revealed a micellar size of 4.1 nm. Subsequent removal of cholate (from an initial concentration of 2% to less than 0.02%) in the presence of Azolectin (soybean lipid extract) led to reconstituted material that could be retained on glass fibre filters. Electron microscopy revealed apparent micelles of approximately 1 μm diameter. Over 16% of the original, membrane-bound, binding sites were recovered in the reconstituted material along with a similar percentage of the protein. This reconstituted material was moderately stable for up to 3 days after its preparation. © 1984.