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

S. Benyhe, A. Ketevan, J. Simon, J. Hepp, K. Medzihradszky, A. Borsodi

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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))-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.

Original languageEnglish
Pages (from-to)52-59
Number of pages8
JournalNeuropeptides
Volume31
Issue number1
DOIs
Publication statusPublished - Jan 1 1997

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ASJC Scopus subject areas

  • Endocrinology
  • Neurology
  • Endocrine and Autonomic Systems
  • Cellular and Molecular Neuroscience

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