title: Affinity labelling of frog brain opioid receptors by dynorphin(1-10) chloromethyl ketone.
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 kappa-opioid binding sites labelled by [3H]ethylketocyclazocine (Ki is approximately equal to 200 nM), whereas its binding to the 1 ([3H]dihydromorphine) and to the delta 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 microM DynCMK reveals a significant decrease in the number of specific binding sites for [3H]ethylketocyclazocine compared to the control values. The kappa-preferring binding properties of the compound suggest that it could serve as an affinity label for the kappa-type of opioid receptors.
authors.affiliation: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged.
authors.forename: S
authors.initials: S
authors.lastname: Benyhe
authors.forename: A
authors.initials: A
authors.lastname: Ketevan
authors.forename: J
authors.initials: J
authors.lastname: Simon
authors.forename: J
authors.initials: J
authors.lastname: Hepp
authors.forename: K
authors.initials: K
authors.lastname: Medzihradszky
authors.forename: A
authors.initials: A
authors.lastname: Borsodi
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology; D000590:Amino Acid Chloromethyl Ketones / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D001923:Brain Chemistry / Q000502:physiology*; D004090:Dihydromorphine / Q000494:pharmacology; D004399:Dynorphins / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D016308:Enkephalin, Leucine-2-Alanine / Q000494:pharmacology; D016314:Ethylketocyclazocine / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011869:Radioligand Assay; D011893:Rana esculenta; D011957:Receptors, Opioid / Q000032:analysis* / Q000378:metabolism; D017473:Receptors, Opioid, kappa / Q000819:agonists; D014316:Tritium
chemical_list: D000345:Affinity Labels; D000590:Amino Acid Chloromethyl Ketones; D000701:Analgesics, Opioid; D008565:Membrane Proteins; D010446:Peptide Fragments; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D014316:Tritium; D016314:Ethylketocyclazocine; D016308:Enkephalin, Leucine-2-Alanine; D004399:Dynorphins; C028557:tyrosyl-alanyl-glycyl-phenylalanyl-leucine chloromethyl ketone; C039666:dynorphin amide (1-10); D004090:Dihydromorphine