title: Met5-enkephalin-Arg6-Phe7, an endogenous neuropeptide, binds to multiple opioid and nonopioid sites in rat brain.
abstract: 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.
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary. benyhe@everx.szbk.uszeged.hu
authors.forename: S
authors.initials: S
authors.lastname: Benyhe
authors.forename: J
authors.initials: J
authors.lastname: Farkas
authors.forename: G
authors.initials: G
authors.lastname: Tóth
authors.forename: M
authors.initials: M
authors.lastname: Wollemann
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism; D019835:FMRFamide; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D008566:Membranes / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D051381:Rats; D011957:Receptors, Opioid / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology
chemical_list: D009479:Neuropeptides; D011957:Receptors, Opioid; D006165:Guanylyl Imidodiphosphate; D004744:Enkephalin, Methionine; D019835:FMRFamide; C027511:enkephalin-Met, Arg(6)-Phe(7)-; D012964:Sodium