title: Synthesis, radiolabeling and receptor binding of [3H][(1S,2R)ACPC2]endomorphin-2.
abstract: Previously, we have shown that substitution of Pro(2) for cis-2-aminocyclopentanecarboxylic acid, ACPC in endomorphin-2 results in an analogue with greatly augmented proteolytic stability, high mu-opioid receptor affinity and selectivity. We now report the synthesis and biochemical characterization of [(3)H][(1S,2R)ACPC(2)]endomorphin-2 with a specific activity of 1.41 TBq/mmol (38.17 Ci/mmol). Specific binding of [(3)H][(1S,2R)ACPC(2)]endomorphin-2 was saturable and of high affinity with an equilibrium dissociation constant, K(d)=1.80+/-0.21nM and receptor density, B(max)=345+/-27 fmol x mg protein(-1) at 25 degrees C in rat brain membranes. Similar affinity values were obtained in kinetic and displacement assays. Both Na(+) and Gpp(NH)p decreased the affinity proving the agonist character of the radioligand. [(3)H][(1S,2R)ACPC(2)]endomorphin-2 retained the mu-specificity of the parent peptide. The new radioligand will be a useful tool to map the topographical requirements of mu-opioid peptide binding due to its high affinity, selectivity and enzymatic stability.
authors.affiliation: Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Attila
authors.initials: A
authors.lastname: Keresztes
authors.forename: Géza
authors.initials: G
authors.lastname: Tóth
authors.forename: Ferenc
authors.initials: F
authors.lastname: Fülöp
authors.forename: Mária
authors.initials: M
authors.lastname: Szucs
mesh_terms: D000818:Animals; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D017450:Receptors, Opioid, mu / Q000378:metabolism*; D013194:Staining and Labeling; D014316:Tritium
chemical_list: D009842:Oligopeptides; D017450:Receptors, Opioid, mu; D014316:Tritium; C105294:endomorphin 2