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 κ-opioid binding sites labelled by [8H]ethylketocyclazocine (Ki ~ 200 nM), whereas its binding to the μ ([3H]dihydromorphine) and to the δ 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 μM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]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.
ASJC Scopus subject areas
- Endocrine and Autonomic Systems
- Cellular and Molecular Neuroscience