The S-3-nitro-2-pyridinesulfenyl (SNpys) group in an affinity ligand can bind to a free thiol group of a cysteine residue in a target receptor molecule, forming a disulfide bond via the thiol-disulfide exchange reaction. SNpys-containing Leu-enkephalin analogues of [D-Ala2, Leu 5]-enkephalyl-Cys(Npys)6 and [D-Ala2, Leu(CH2SNpys)5]enkephalin, and dynorphin A analogues of [D-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide and [D-Ala2,Cys(Npys)8]dynorphin A-(1-9) amide have been found to affinity-label all of the δ, μ (rat brain), and κ (guinea pig brain) opioid receptor subtypes. In this study, using these chemically synthesized SNpys-containing analogues, we attempted to identify the analogues that affinity-label the cysteine residue at position 60 of the δ opioid receptor. We first established the assay procedure, principally based on the receptor binding assay to use COS-7 cells expressing the δ opioid receptor. Then, using a mutant δ receptor with the Cys60→Ala substitution, we assayed the SNpys-containing analogues for their specific affinity-labeling. [D-Ala2,Cys(Npys)12]dynorphin A-(1-13) amide was found to have drastically reduced labeling activity for this mutant receptor as compared to its activity for the wild-type δ receptor. Other analogues exhibited almost the same activity for both the wild-type and mutant δ receptors. These results indicate that the δ-Cys60 residue has a free thiol group, which is labeled by [D-Ala2,Cys(Npys) 12]dynorphin A-(1-13) amide.
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