digitonin and bremazocine

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).

Topics: Analgesics; Animals; Benzomorphans; Cell Membrane; Centrifugation, Density Gradient; Cerebellum; Chromatography, Gel; Digitonin; Guinea Pigs; In Vitro Techniques; Receptors, Opioid; Receptors, Opioid, kappa; Sucrose

Solubilization of opioid binding sites from guinea pig cerebellum by digitonin, in the absence and presence of NaCl, resulted in very similar yields (25-30%) of [3H]bremazocine binding. Saturation curves of [3H]bremazocine binding give linear Scatchard plots for both soluble and membrane-bound binding sites yielding similar Kd's and Bmax's. Soluble kappa sites seem to resemble closely their membrane-bound counterparts and retain high affinity and selectivity for various kappa opioid ligands. The apparent molecular weight of soluble kappa sites is ca. 4 X 10(5). Results from this study, along with our previous findings with toad and guinea pig brain, indicate that kappa sites (unlike mu and delta) can be solubilized in good yield by digitonin even in the absence of NaCl. This supports the hypothesis that kappa sites may represent molecular species different from those of mu and delta sites.

Topics: Animals; Benzomorphans; Cerebellum; Digitonin; Guinea Pigs; In Vitro Techniques; Kinetics; Molecular Weight; Receptors, Opioid; Receptors, Opioid, kappa; Solubility