14.beta.-(Bromoacetamido)morphine irreversibly labels .mu. opioid receptors in rat brain membranes

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Abstract
The binding properties of 14.beta.-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM [3H][D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the .mu. binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The .mu. receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the .delta.-selective peptide [3H][D-penicillamine2,D-penicillamine5]enkephalin (DPDPE) and (-)-[3H]bremazocine in the presence of .mu. and .delta. blockers, selective for .kappa. binding sites. Under conditions where 90% of the 0.25 nM [3H]DAGO binding sites were blocked, 80% of the 0.8 nM [3H]naloxone binding and 50% of the 0.25 nM 125I-labeled .beta.h-endorphin binding were inhibited by BAM alkylation. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the .mu. site did not afford protection. These studies have demonstrated that when a disulfide bond at or near .mu. opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of .mu. opioid receptors.