Opiate receptors from different tissue sources: solubilization and characterization.

Abstract

Abstract: Membrane‐bound opiate receptors from neuroblastoma‐glioma hybrid cells and from different parts of the rat brain (whole brain minus cerebellum, cortex, thalamus‐hypothalamus and cerebellum) were labeled with the methionine‐enkephalin analogue, D‐[3H]Ala2‐Met‐enkephalinamide, and solubilized with the nonionic detergent Brij 36T. The protease inhibitors bacitracin, phenylmethylsulfonyl fluoride, Trasylol, and leupeptin were included in the solubilization buffer to minimize proteolysis. Two simple techniques, ammonium sulfate precipitation and activated charcoal absorbence, were adapted to separate the free and the macromolecule‐bound ligands. The solubilized receptor‐[3H]enkephalin complexes were partially purified by consecutive passages through Sephadex G‐75 and Sepharose 6B columns. Of the three peaks of radioactivity that were observed in the effluent of the Sepharose column, two contained proteins, and one of them, with a Stokes radius of 59 Å, seemed to contain the specific opiate receptor, as evidenced by additional experiments. This peak was further purified on thiol‐Sepharose or diethylaminoethanol‐Sephadex columns that were eluted with a gradient of 0–50 mM dithiothreitol or with 1.0 M KCI, respectively. The receptor‐[3H]enkephalin complex from neuroblastoma‐glioma cells (apparent δ‐type receptors) binds less to the thiol‐Sepharose beads than receptor‐(3H]enkephalin prepared from the hypothalamus‐thalamus, which is rich in μ receptors. The [3H]enkephalin receptor complexes of the various sources also differed in their stability. The dissociation of the ligand from the neuroblastoma‐glioma receptor was monophasic, with a half‐ life of 250 min, whereas that of two brain regions was biphasic, with half‐lives of 195–330 min and 10,000 min. The methods described may be of use for further purification of soluble opiate receptors, either active or cross‐linked to the ligand.