dihydromorphine and tyrosyl-alanyl-glycyl-phenylalanyl-leucine-chloromethyl-ketone

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)-Gly11 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 binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta sites ([3H]D-Ala2-Leu5]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 [3H]ethylketocyclazocine binding sites. Saturation binding analysis of the membranes preincubated with 50 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine 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.

Topics: Affinity Labels; Amino Acid Chloromethyl Ketones; Analgesics, Opioid; Animals; Binding, Competitive; Brain Chemistry; Dihydromorphine; Dynorphins; Enkephalin, Leucine-2-Alanine; Ethylketocyclazocine; Membrane Proteins; Peptide Fragments; Radioligand Assay; Rana esculenta; Receptors, Opioid; Receptors, Opioid, kappa; Tritium