Characterization of rat brain opioid receptors by [Tyr-3,5-3H]1, D-Ala2, Leu5-enkephalin binding

[Tyr-3,5-3H]1, D-Ala2, Leu5-enkephalin [( 3H]DALA) was used for labeling the opioid receptors of rat brain plasma membranes. The labeled ligand was prepared from [Tyr-3,5-diiodo]1, D-Ala2, Leu5-enkephalin by catalytic reductive dehalogenation in the presence of Pd catalyst. The resulting [Tyr-3,5-3H]1, D-Ala2, Leu5-enkephalin had a specific activity of 37.3 Ci/mmol. In the binding experiments steady-state level was reached at 24 degrees C within 45 min. The pseudo first order association rate constant was 0.1 min-1. The dissociation of the receptor-ligand complex was biphasic with k-1-s of 0.009 and 0.025 min-1. The existence of two binding sites was proved by equilibrium studies. The high affinity site showed a KD = 0.7 nM and Bmax = 60 fmol/mg protein; the low affinity site had a KD = 5 nM and Bmax = 160 fmol/mg protein. A series of opioid peptides inhibited [3H]DALA binding more efficiently than morphine-like drugs suggesting that labeled ligand binds preferentially to the delta subtype of opioid receptors. Modification of the original peptides either at the C or N terminal ends of the molecules resulted in a decrease in their affinity.