caa a22 4500 397556748 CHVBK 20180308164804.0 cr unu---uuuuu 161202e199608 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021433 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021433 Reversible Affinity Labeling of Opioid Receptors via Disulfide Bonding: Discriminative Labeling of μ and δ Subtypes by Chemically Activated Thiol-Containing Enkephalin Analogs [Elektronische Daten] [Teruo Yasunaga, Shihoko Motoyama, Takeru Nose, Hiroaki Kodama, Michio Kondo, Yasuyuki Shimohigashi] The 3-nitro-2-pyridinesulfenyl (Npys) group bound to a mercapto group is a highly activated electrophilic reagent, which only reacts with a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. We incorporated the Npys group into enkephalin analogs to affinity label μ and δ opioid receptors. When rat brain membranes were incubated with [D-Ala2,Leu(CH2SNpys)5]enkephalin, and assayed for the inhibition of binding of DAGO and DSLET enkephalin analogs to opioid receptors, the number of receptors decreased sharply, depending upon the concentration of this SNpyscontaining enkephalin. It was found that this enkephalin analog occupies μ receptors highly specifically (EC50=51 nM) and almost 100 times more selectively than δ receptors. In contrast, [D-Ala2,Leu5]enkephalyl-Cys(Npys)6 attached covalently to δ receptors (ECao50= nM) about 150 times more selectively than to μ receptors. Although N-ethylmaleimide also inhibited the binding of DAGO and DSLET, four to six orders of magnitude higher concentrations were required as compared to SNpys-containing enkephalins. When enkephalin- bound rat membranes were treated with dithiothreitol, the loss of receptors was reversed, depending upon the concentration of and incubation time with dithiothreitol. The recovery was much faster (about 1,000 times) for δ receptors than for μ receptors. The present results indicated that both μ and δ receptors in rat brain consist of a free mercapto group near the enkephalin binding site and that SNpys-containing enkephalins can label these mercapto groups discriminatively. The disulfide bond between [D-Ala2,Leu5]enkephalyl- Cys6 and δ receptors appears to be exposed, while that between [D-Ala2,Leu(CH2-SNpy8)]enkephalin and μ receptors is shielded. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence activated thiol nationallicence affinity labeling nationallicence disulfide bond nationallicence enkephalins nationallicence opioid receptors nationallicence Yasunaga Teruo Manufacturing Process Development Division, Otsuka Pharmaceutical Co., Ltd. Saga Factory, Kanzaki Saga 842-01 aut Motoyama Shihoko Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University Fukuoka 812-81 aut Nose Takeru Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University Fukuoka 812-81 aut Kodama Hiroaki Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840 Saga 840 aut Kondo Michio Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840 Saga 840 aut Shimohigashi Yasuyuki Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University Fukuoka 812-81 aut The Journal of Biochemistry Oxford University Press 120/2(1996-08), 459-465 0021-924X 120:2<459 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021433 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021433 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Yasunaga Teruo Manufacturing Process Development Division, Otsuka Pharmaceutical Co., Ltd. Saga Factory, Kanzaki Saga 842-01 aut NATIONALLICENCE 700 1- Motoyama Shihoko Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University Fukuoka 812-81 aut NATIONALLICENCE 700 1- Nose Takeru Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University Fukuoka 812-81 aut NATIONALLICENCE 700 1- Kodama Hiroaki Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840 Saga 840 aut NATIONALLICENCE 700 1- Kondo Michio Department of Chemistry, Faculty of Science and Engineering, Saga University, Saga 840 Saga 840 aut NATIONALLICENCE 700 1- Shimohigashi Yasuyuki Laboratory of Biochemistry, Department of Chemistry, Faculty of Science, Kyushu University Fukuoka 812-81 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/2(1996-08), 459-465 0021-924X 120:2<459 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford