Discriminative disulfide-bonding affinity labeling of opioid receptor subtypes.
The affinity-labeling technique is an extremely important method in receptor biochemistry. The 3-nitro-2-pyridinesulfenyl (Npys) group, attached to a mercapto group, can react only with a free thiol group (the beta-mercapto group of cysteine residue) of the target receptor molecules, forming a disulfide bond. This disulfide bonding is mediated through the thiol-disulfide exchange reaction. Unlike other labeling methods, the approach utilizing such chemically activated thiol-containing ligands is able to reproduce an unlabeled protein by treatment with dithiothreitol, a reducing reagent. This provides several unique aspects for the studies elucidating the structure-function relationships between the peptide and the receptor. Based on the SNpys affinity technique, we have achieved the discriminative disulfide-bonding affinity labeling of the three different subtypes of opioid receptors: mu, delta and kappa. This article reviews our novel affinity techniques in the in vitro receptor biochemistry.
Shirasu
N
N
Laboratory of Structure-Function Biochemistry, Department of Chemistry, Faculty and Graduate School of Sciences, Kyushu University, Fukuoka 812-8581, Japan.
Shimohigashi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D003198:Computer Simulation; D003545:Cysteine; D004220:Disulfides / Q000032:analysis*; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000493:pharmacokinetics; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D011957:Receptors, Opioid / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D004220:Disulfides; D008024:Ligands; D011957:Receptors, Opioid; D011994:Recombinant Proteins; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D003545:Cysteine