Affinity labeling of mu opioid receptors by sulfhydryl alkylating derivatives of morphine and morphinone
Bidlack, J.M.; Frey, D.K.; Kaplan, R.A.; Seyed-Mozaffari, A.; Archer, S.
Molecular Pharmacology 37(1): 50-59
1990
ISSN/ISBN: 0026-895X PMID: 1688995 Document Number: 353854
After reduction of a disulfide bond at or near the .mu. opioid binding site in rat brain membranes, incubating membranes with 14.beta.-bromoacetamido derivatives of either morphine, dihydromorphine, morphinone, or dihydromorphinone resulted in the irreversible inhibition of .mu. opioid binding to rat brain membranes. Without the addition of the disulfide bond-reducing reagent dithiothreitol, these affinity ligands bound reversibly to opioid binding sites. Binding to either .delta. or .kappa. opioid binding sites was not altered by alkylation of the membranes with the affinity ligands. The percentage of irreversible inhibition of .mu. opioid binding was dependent on the time and temperature of the incubation of membranes with the affinity ligands and on the concentrations of dithiothreitol and the affinity ligands. Incubating membranes with morphine afforded almost complete protection from alkylation of the .mu. opioid binding site. Naloxone and the l-isomer levorphanol also protected the site from alkylation, whereas the d-isomer dextrorphan and the .kappa.-selective opioid U50,488H did not protect the site. The .mu.-selective peptide [D-Ala2,(Me)Phe4,Gly(ol)5]enkephalin was the peptide that afforded the greatest protection. These studies have shown that, after the reduction of a disulfide bond at or near the .mu. opioid binding site, this sulfhydryl group can be specifically alkylated, resulting in the affinity labeling of the .mu. opioid binding site.