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Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and nonopioid sites in rat brain.

  • Benyhe, S
  • Farkas, J
  • Tóth, G
  • Wollemann, M
Published Article
Journal of neuroscience research
Publication Date
May 01, 1997
PMID: 9160247


Receptor binding properties of the naturally occurring opioid heptapeptide MERF were studied in rat brain membrane preparations using tritium-labeled derivative of the peptide with 40 Ci/mmol specific radioactivity. Binding assays were performed in the presence of broad-spectrum peptidase inhibitors at 0 degree C. Under these conditions, the equilibrium binding was achieved in 30-40 min, and approximately 90% of the applied radioligand remained unchanged as determined by HPLC analysis. The apparent affinity (Kd value) of [3H]Met-enkephalin-Arg6-Phe7, calculated from saturation binding data, was 10.2 +/- 2.5 nM, and the maximal number (Bmax) of the heptapeptide binding sites was found to be 468 +/- 43 fmol/mg protein. About half the sites represent nonopioid sites because the Bmax was only 255 +/- 30 fmol/mg, when the nonspecific binding was measured with 1 microM naloxone. The rank order potencies of the examined compounds revealed that the opioid component of [3H]Met-enkephalin-Arg6-Phe7 recognition site are probably not mu and certainly not kappa 1 sites, whereas these sites are characterized by a kappa 2-like binding profile. Considering the discrepancies between rat and frog brain found in the affinity of some compounds, including naltrindole and norbinaltorphimine, the presence of a novel, MERF-selective "heptapeptide" binding site in rat brain membranes is also suggested. A number of the heterologous competition curves could be described by a high-affinity stereospecific component and a substantially lower-affinity binding element, which could completely be displaced with several peptide ligands such as Met5-enkephalin, dynorphin(1-13), and unlabeled MERF but not by other compounds such as [D-Ala2-(Me)Phe4-Gly5-ol]enkephalin, morphine, or naloxone. [3H]Met-enkephalin-Arg6-Phe7 binding can also be inhibited by FMRF-amide analogs and sigma receptor ligands, such as (+)N-allyl-normetazocine and haloperidol, although with moderate affinity. It is concluded that the stereospecific high-affinity binding is of opioid in character, whereas the residual sites characterized with their lower affinity are naloxone-insensitive nonopioid sites.

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