Separation of κ‐opioid receptor subtype from frog brain

Complete separation of the [ 3 H]ethylketocyclazocine ([ 3 H]EKC) specific binding (k subtype) from tritiated Tyr‐D‐Ala 2 ‐Me‐Phe 4 ‐Gly‐ol 5 enkephalin (DAGO) and Tyr‐D‐Ala 2 ‐L‐Leu 5 ‐enkephalin (DALA) binding (μ‐and δ‐subtypes, respectively) was achieved by Sepharose‐6B chromatography and sucrose density gradient centrifugation of digitonin solubilized frog brain membranes. The apparent sedimentation coefficient ( S 20 ,w) for the k receptor‐detergent complex was 13.1 S and the corresponding Stokes radius 64 Å. The isolated fractions exhibited high affinity for EKC and bremazocine, whereas μ‐ and δ‐specific ligands were unable to compete for the [ 3 H]EKC binding sites, indicating that the κ subtype represents a separate molecular entity from the μ and δ receptor sites.