Solubilization and characterization of the kappa-opioid receptor type from guinea-pig cerebellum

The kappa-opioid receptor (kappa Op) from guinea-pig cerebellum membranes has been solubilized in an active form and in good yield (30-40%) with digitonin in 10 mM Tes-KOH buffer, pH 7.4. [3H]Bremazocine (KD = 1.1 nM in the soluble extract) was used to monitor the binding and hydrodynamic characteristics of the digitonin-solubilized receptor, s-kappa Op. Opioid agonists and antagonists bind s-kappa Op with a generally lower (yet still high) apparent affinity than they do its membrane-bound counterpart, m-kappa Op, but with the same rank order of potency. In particular, U50488, a kappa selective agonist, binds s-kappa Op with a considerably higher apparent affinity than do [D-Ala2, MePhe4, Glyol5]enkephalin, a mu selective agonist, and [D-Thr2,Leu5]enkephalyl-Thr, a delta selective agonist. s-kappa Op has also retained substantial stereospecificity since levorphanol was nearly 200-fold as potent as dextrorphan in competing with binding of [3H]bremazocine in the soluble extract. However, s-kappa Op appeared to be desensitized to regulation by Na+ ions: the selective inhibition by the allosteric effector of binding of agonists at the m-kappa Op was no longer observed in digitonin extracts from guinea-pig cerebellum membranes. The s-kappa Op behaved as a unique macromolecular entity both in molecular exclusion chromatography (appr. rs = 6.7 nm) and in sedimentation on linear density gradients (app. S20,w = 11.8 S).