AFFINITY LABELING OF FROG BRAIN OPIOID RECEPTORS BY DYNORPHIN((1-10))CHLOROMETHYL KETONE

It has been previously found that chloromethyl ketone derivatives of e nkephalins bind irreversibly to the opioid receptors in vitro. Recentl y a novel affinity reagent, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Gl y 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 kappa-opioid bi nding sites labelled by [H-3]ethylketocyclazocine (Ki approximate to 2 00 nN), whereas its binding to the mu ([H-3]dihydromorphine) and to th e delta sites ([H-3]D-Ala(2)-Leu(5)]enkephalin) is weaker. Preincubati on of the frog brain membranes with DynCMK at micromolar concentration s results in a washing-resistant and dose-dependent inhibition of the [H-3]ethylketocyclazocine binding sites. Saturation binding analysis o f the membranes preincubated with 50 mu M DynCMK reveals a significant decrease in the number of specific binding sites for [H-3]ethylketocy clazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.