title: Opiorphin highly improves the specific binding and affinity of MERF and MEGY to rat brain opioid receptors.
abstract: Endogenously occurring opioid peptides are rapidly metabolized by different ectopeptidases. Human opiorphin is a recently discovered natural inhibitor of the enkephalin-inactivating neutral endopeptidase (NEP) and aminopeptidase-N (AP-N) (Wisner et al., 2006). To date, in vitro receptor binding experiments must be performed either in the presence of a mixture of peptidase inhibitors and/or at low temperatures, to block peptidase activity. Here we demonstrate that, compared to classic inhibitor cocktails, opiorphin dramatically increases the binding of [(3)H]MERF and [(3)H]MEGY ligands to rat brain membrane preparations. We found that at 0 °C the increase in specific binding is as high as 40-60% and at 24 °C this rise was even higher. In contrast, the binding of the control [(3)H]endomorphin-1, which is relatively slowly degraded in rat brain membrane preparations, was not enhanced by opiorphin compared to other inhibitors. In addition, in homologous binding displacement experiments, the IC(50) affinity values measured at 24 °C were also significantly improved using opiorphin compared to the inhibitor cocktail. In heterologous binding experiments the differences were less obvious, but still pronounced using [(3)H]MERF and MEGY compared to dynorphin(1-11), or naloxone and DAGO competitor ligands.
authors: Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, P.O.B. 521, H-6701, Szeged, Hungary.
authors: Fanni
authors: F
authors: Tóth
authors: Géza
authors: G
authors: Tóth
authors: Sándor
authors: S
authors: Benyhe
authors: Catherine
authors: C
authors: Rougeot
authors: Mária
authors: M
authors: Wollemann
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004744:Enkephalin, Methionine / Q000031:analogs & derivatives* / Q000378:metabolism; D008297:Male; D009842:Oligopeptides / Q000494:pharmacology* / Q000502:physiology; D011480:Protease Inhibitors / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000378:metabolism*; D012471:Salivary Proteins and Peptides / Q000494:pharmacology* / Q000502:physiology
chemical_list: C504157:Met-enkephalin-glycyl-tyrosine; D009842:Oligopeptides; D011480:Protease Inhibitors; D011957:Receptors, Opioid; D012471:Salivary Proteins and Peptides; C105293:endomorphin 1; C517879:glutaminyl-arginyl-phenylalanyl-seryl-arginine; D004744:Enkephalin, Methionine; C027511:enkephalin-Met, Arg(6)-Phe(7)-