Characterization of [3H]Met-enkephalin-Arg6-Phe7 binding to opioid receptors in frog brain membrane preparations

Mária Wollemann, Judit Farkas, Géza Tóth, Sándor Benyhe

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27 Citations (Scopus)

Abstract

A tritiated heptapeptide, [3H]Tyr-Gly-Gly-Phe-Met-Arg-Phe ([3H]Met- enkephalin-Arg6-Phe7), with high specific radioactivity has been synthesized in order to characterize its opioid binding activity to frog brain membrane fractions. The apparent K(D) value of the radioligand calculated from homologous displacement experiments was 3.4 nM, and the maximal number of specific binding sites was 630 fmol/mg of protein. The K(D) determined from equilibrium saturation binding studies was found to be 3.6 nM. However, the Hill coefficient was far below unity (n(H) = 0.43), which suggests the presence of a second, lower affinity binding site. The presence of this binding component is strengthened by the displacement experiments performed with levorphanol and some other ligands. It is assumed that the lower affinity site has no opiate character. The rank order of potency of the applied ligands in competing reversibly with [3H]Met-enkephalin-Arg6-Phe7 binding reflects a κ2- and/or δ-subtype specificity of the heptapeptide. Binding to a κ1 and/or μ site of opioid receptors is excluded, but the existence of a novel endogenous opiate receptor subtype for Met-enkephalin- Arg6-Phe7 in frogs cannot be ruled out. The [3H]-Met-enkephalin-Arg6- Phe7 binding was inhibited by both sodium ions and GppNHp, which suggests the opioid agonist character of the heptapeptide.

Original languageEnglish
Pages (from-to)1460-1465
Number of pages6
JournalJournal of neurochemistry
Volume63
Issue number4
Publication statusPublished - Oct 1 1994

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Keywords

  • Frog brain
  • Met- enkephalin-Arg-Phe
  • Opioid receptor subtypes
  • Radioligand binding
  • Tritium labelling
  • κ site

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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