Pharmacology of a new tritiated endomorphin-2 analog containing the proline mimetic cis-2-aminocyclohexanecarboxylic acid |
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Authors: | Keresztes Attila Birkás Erika Páhi Annamária Tóth Géza Bakota Lidia Gulya Károly Szücs Mária |
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Institution: | a Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary b Department of Cell Biology and Molecular Medicine, Faculty of Medicine and Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary |
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Abstract: | As part of ongoing work aimed at generating proteolytically stable, readily applicable, radiolabeled endomorphin-2 (EM-2) analogs for elucidation of the topological requirements of peptide binding to μ-opioid receptors, we report here on the synthesis, radiolabeling, binding kinetics and binding site distribution of an EM-2 analog in which Pro2 is replaced by 2-aminocyclohexanecarboxylic acid, ACHC. 3H](1S,2R)ACHC]2EM-2 (specific activity 63.49 Ci × mmol−1) bound specifically to its binding sites with high affinity (KD = 0.55 ± 0.06 nM) and saturably, yielding a receptor density, Bmax of 151 ± 4 fmol × mg protein−1 in rat brain membranes. A similar affinity value was obtained in kinetic assays. Both Na+ and Gpp(NH)p decreased the affinity, proving the agonist character of the radioligand. Specific μ-opioid ligands displaced the radioligand with much higher affinities than did δ- and κ-ligands. The autoradiographic distribution of the binding sites of 3H](1S,2R)ACHC]2EM-2 agreed well with the known locations of the μ-opioid receptors in the rat brain. In consequence of its high affinity, selectivity and enzymatic resistance 19], the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding. |
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Keywords: | Endomorphin Peptide synthesis 2-Aminocylcohexanecarboxylic acid (ACHC) Radiolabeling Receptor binding Autoradiography Proteolytic stability |
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