Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues

Kaname Isozaki, Hidehiko Fukahori, Takeshi Honda, Naoto Shirasu, Kazushi Okada, Takeru Nose, Kazuyasu Sakaguchi, Yasuyuki Shimohigashi

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)511-522
Number of pages12
JournalLetters in Peptide Science
Volume10
Issue number5-6
DOIs
Publication statusPublished - Nov 1 2003

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Dynorphins
delta Opioid Receptor
Enkephalins
Amides
Labeling
Labels
Assays
Brain
Opioid Receptors
Affinity Labels
Sulfhydryl Compounds
Leucine Enkephalin
Rats
Disulfides
Substitution reactions
Cysteine
Ligands
Cells
Molecules
3-nitro-2-pyridinesulfenyl

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues. / Isozaki, Kaname; Fukahori, Hidehiko; Honda, Takeshi; Shirasu, Naoto; Okada, Kazushi; Nose, Takeru; Sakaguchi, Kazuyasu; Shimohigashi, Yasuyuki.

In: Letters in Peptide Science, Vol. 10, No. 5-6, 01.11.2003, p. 511-522.

Research output: Contribution to journalArticle

Isozaki, K, Fukahori, H, Honda, T, Shirasu, N, Okada, K, Nose, T, Sakaguchi, K & Shimohigashi, Y 2003, 'Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues', Letters in Peptide Science, vol. 10, no. 5-6, pp. 511-522. https://doi.org/10.1007/BF02442583
Isozaki, Kaname ; Fukahori, Hidehiko ; Honda, Takeshi ; Shirasu, Naoto ; Okada, Kazushi ; Nose, Takeru ; Sakaguchi, Kazuyasu ; Shimohigashi, Yasuyuki. / Site-directed affinity-labeling of delta opioid receptors by SNpys-containing enkephalin and dynorphin analogues. In: Letters in Peptide Science. 2003 ; Vol. 10, No. 5-6. pp. 511-522.
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