BIOCHEMICAL-CHARACTERIZATION OF HIGH-AFFINITY H-3 OPIOID BINDING - FURTHER EVIDENCE FOR MU1-SITES

Abstract

In saturation studies with [3H]dihydromorphine, unlabeled D-Ala2-D-Leu5-enkephalin (1 nM) inhibited the high-affinity binding component far more potently than the lower-affinity one. Morphine (1 nM) inhibited the higher-affinity binding of 3H-D-Ala2-D-Leu5-enkephalin to a greater extent than its lower-affinity binding component, consistent with a common high-affinity binding site for opiates and enkephalins. Treatment of tissue [rat brain membranes] with either trypsin (1 .mu.g/ml) or N-ethylmaleimide (25 .mu.M) effectively eliminated the high-affinity binding component of a series of 3H-opiates and opioid peptides. Competition studies following both treatments were consistent with a common high-affinity binding site. Both treatments eliminated the ability of low morphine concentrations (< nM) to inhibit 3H-D-Ala2-D-Leu5-enkephalin binding and of low D-Ala2-D-Leu5-enkephalin concentrations (< nM) to inhibit [3H]dihydromorphine binding. Protection experiments examining N-ethylmaleimide (25 .mu.M) inhibition of [3H]dihydromorphine binding showed significant protection (P < 0.002) by both unlabeled D-Ala2-D-Leu5-enkephalin and morphine (both at 1 nM). When studied together, both naloxonazine and N-ethylmaleimide inhibited [3H]dihydromorphine binding to a similar extent. Tissue previously treated with naloxonazine was far less sensitive to N-ethylmaleimide than was untreated control tissue, consistent with the possibility that both treatments affected the same site. The concept a common high-affinity binding site for opiates and opioid peptides is supported.