1989 Fiscal Year Final Research Report Summary
Purification of opioid receptors and preparation and application of monoclonal antibodies to them
| Project/Area Number |
63480464
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
SATOH Masamichi Kyoto University, Faculty of Pharmaceutical Scineces, Professor, 薬学部, 教授 (80025709)
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| Co-Investigator(Kenkyū-buntansha) |
UEDA Hiroshi Kyoto Univ., Fac. of Pharm. Sci., Instructor, 薬学部, 助手 (00145674)
KURAISHI Yasushi Kyoto Univ., Fac. of Pharm. Sci., Assoc.Prof., 薬学部, 助教授 (80111970)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Opioid Receptor / Monoclonal Antibody / NG 108-15 Cell / Signal Transduction |
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| Research Abstract |
Opioid receptors are classified into three different subtypes, g,&, and K.
Opioid receptors are classified into three different subtypes, mu,delta, and kappa. However, little is known of molecular basis of mechanisms of pharmacological actions of opioids through such different subtypes. The present works were focused on the preparation of monoclonal antibodies to opioid receptors and application in order to clarify such molecular mechanisms. Neuroblastoma x glioma hybrid NG108-15 cell membranes were immunized into BALB/C mice for 4 months. The spleen cells were fused with myeloma cells and the culture supernatants in wells containing hybridomas were used for assays in delta-opioid binding experiments. delta-OpR-l showed not only a potent inhibition of delta-opioid agonist binding with a decrease in its Bmax value, but also a blocking of delta-agonist-induced inhibition of adenylate cyclase activity. On the other hand, delta-OpR-3 showed an inhibition of delta-opioid agonist binding with a decrease in its affinity. This antibody was found to stain immunocytochemically delta-opioid receptor expressed in NG108-15 cells. These monoclonal antibodies will be useful for purification of delta-opioid receptors or its expression cloning by immunoscreening methods.
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[Publications] Ueda,H.,Harada,H.,Nozaki,M.,Katada,T.,Ui,M.,Satoh,M.and Takagi,H.: "Reconstitution of vat brain μ-opioid receptors with purified guanine nucleotide-binding regulatory protains,Gi and Go." Proc.Natl.Acad.Sci.USA. 85. 7013-7017 (1988)
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「研究成果報告書概要(和文)」より
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[Publications] Harada,H.,Ueda,H.,Katada,T.,Ui,M.,and Satoh,M.: "Phosphorylation of μ-opioid receptor-a putative inedarism of selective uncoupling of receptor-Gi interaction,measured with low-km GTP ase and guanine nucleotide-sensitive binding" Neurosci.Lett.100. 221-226 (1989)
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「研究成果報告書概要(和文)」より
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[Publications] Ueda,H.,Misawa,H.,Katada,T.,Ui,M.,Takagi,H.and Satoh,M.: "Functional reconstitution of purified Gi and Go with μ-opioid receptors in quinea pig striatal membranes pre-treated with micromolar concentrations of N-ethylinaleimide" J.Neurochem.(in press). (1990)
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[Publications] Ueda, H., Harada, H., Nozaki, M., Katada, T., Ui, M., Satoh, M. and Takagi, H.: "Reconstitution of rat brain mu -opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go." Proc. Natl. Acad. Sci. USA, 85, 7013-7017 (1988).
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「研究成果報告書概要(欧文)」より
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[Publications] Harada, H., Ueda, H., Wada, Y., Katada, T., Ui, M. and Satoh, M.: "Phosphorylation of mu -opioid receptor - a putative mechanism of selective uncoupling of receptor - Gi interaction, measured with low-Km GTPase and guanine nucleotide-sensitive agonist binding." Neurosci. Lett. 100, 221-226 (1989).
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「研究成果報告書概要(欧文)」より
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[Publications] Ueda, H., Nishimura, K., Ishida, K., Uno, S. and Satoh, M.: "Monoclonal antibody to delta - opioid receptors" Seikagaku, 61, 930 (1989).
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「研究成果報告書概要(欧文)」より
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[Publications] Ueda, H., Misawa, H., Katada, T., Ui, M., Takagi, H. and Satoh, M.: "Functional reconstitution of purified Gi and Go with mu -opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide." J. Neurochem. (1990).
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