Inactivation of the Purified Bovine μ Opioid Receptor by Sulfhydryl Reagents
- Cite this article as:
- Gioannini, T.L., Onoprishvili, I., Hiller, J.M. et al. Neurochem Res (1999) 24: 37. doi:10.1023/A:1020923928936
We have investigated the role of cysteine residues in a highly purified μ opioid receptor protein (μORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of μORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5′-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([3H]DAMGO) was much more sensitive to inactivation than antagonist binding ([3H]bremazocine). Prebinding μORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the μ opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.