Solubilization and characterization of mu, delta, and kappa opioid binding sites from guinea pig brain: physical separation of kappa receptors.

Abstract

Sucrose density gradient centrifugation of digitonin-solubilized opioid binding sites from guinea pig brain and cerebellum was carried out. Centrifugation of extracts of whole brain into a gradient devoid of Na and low in digitonin revealed the presence of 2 well-separated peaks of opioid binding activity. Peak A had the binding characteristics of .kappa. sites, whereas peak B seemed to be a mixture of .mu. and .delta. sites. When extracts of guinea pig cerebellum were treated in the same manner, a single peak of binding activity was obtained that coincided with peak A from guinea pig brain and exhibited the characteristics of .kappa. binding sites. All 3 sites closely resembled their membrane-bound counterparts, retaining good affinity and selectivity for their appropriate ligands. The apparent sedimentation coefficients (s20,w) of the digitonin-solubilized binding sites present in the 2 peaks were 19 s for peak A and 34-39 s for peak B, and the estimated apparent MW were 400,000 for .kappa. sites and 750,000-875,000 for the mixture of .mu. and .delta. sites. Evidently .kappa. sites constituted separate molecular entities from .mu. and .delta. sites.