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°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 aK _D=0.7 nM andB _max=60 fmol/mg protein; the low affinity site had aK _D=5 nM andB _max=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 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.