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 , the new radioligand will be a good tool in studies of the topographical requirements of μ-opioid-specific peptide binding.
- 2-Aminocylcohexanecarboxylic acid (ACHC)
- Peptide synthesis
- Proteolytic stability
- Receptor binding
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience