Covalent labeling of mu opioid binding site by [3H]beta-funaltrexamine.

[3H]beta-funaltrexamine ([3H]beta-FNA) bound irreversibly to bovine striatal membranes. Naloxone inhibited the irreversible binding of 5 nM [3H]beta-FNA in a dose-dependent manner and maximally inhibited this binding at approximately 1 microM. Thus, the specific irreversible binding of [3H]beta-FNA to opioid receptors was defined as that which could be inhibited by 1 microM naloxone. This specific irreversible binding of [3H]beta-FNA was characterized. Exclusion of Na+ from the incubation medium reduced the specific binding of [3H]beta-FNA, and Na+ could be substituted by Li+ but not by K+, Cs+, Mg2+, or guanylylimidodiphosphate. The specific irreversible binding was saturable, time- and temperature-dependent, and was linearly related to tissue mass. Several drugs were used to characterize this specific binding. Levorphanol was 1000 times more potent as an inhibitor than dextrorphan. mu Opioid ligands (sufentanil and morphine) were much better inhibitors than delta (ICI174,864) or kappa (U50,488H) ligands. The potency of [D-Ala2, D-Leu5]enkephalin (DADLE) was between those of sufentanil and ICI174,864. These results demonstrated that under appropriate conditions [3H]beta-FNA specifically and irreversibly bound to the mu opioid binding site. Membrane preparations labeled with [3H]beta-FNA in the presence or absence of 1 microM naloxone or beta-FNA were subjected to polyacrylamide gel electrophoresis under denaturing and reducing conditions. Fluorograms showed that [3H]beta-FNA specifically bound to a protein (most likely the mu opioid binding site) that migrated as a band with a molecular weight range of 68,000-97,000. Such electrophoretic behavior indicates that it is likely to be a glycoprotein. The glycoprotein nature was confirmed by its adsorption onto a wheat germ lectin-Sepharose column after solubilization and subsequent elution by N-acetyl-D-glucosamine. In this lectin column eluate, the mu opioid receptor was the only protein band labeled by [3H]beta-FNA in the total binding preparation, and no labeled protein was observed in the nonspecific binding preparation. When the wheat germ lectin column eluate of the total binding was treated with peptide:N-glycosidase F, the broad labeled band of Mr 68,000-97,000 became a sharp band of Mr 57,000 with high radioactivity and a faintly labeled band of Mr 49,000.