Affinity labelling of frog brain opioid receptors by dynorphin(1–10) chloromethyl ketone

Abstract It has been previously found that chloromethyl ketone derivatives of enkephalins bind irreversibly to the opioid receptors in vitro. Recently a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gly chloromethyl ketone (Dynorphin (1–10) -Gly 11 chloromethyl ketone, DynCMK) was synthesized, and its binding characteristics to frog ( Rana esculenta ) brain membranes were evaluated. In competition experiments, the product shows a relatively high affinity for the κ-opioid binding sites labelled by [ 3 H]ethylketocyclazocine (Ki ≈ 200 nM), whereas its binding to the μ ([ 3 H]dihydromorphine) and to the δ sites ([ 3 H]D-Ala 2 -Leu 5 ]enkephalin) is weaker. Preincubation of the frog brain membranes with DynCMK at micromolar concentrations results in a washing-resistant and dose-dependent inhibition of the [ 3 H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 μM DynCMK reveals a significant decrease in the number of specific binding sites for [ 3 H]ethylketocyclazocine compared to the control values. The κ-preferring binding properties of the compound suggest that it could serve as an affinity label for the κ-type of opioid receptors.