| Project/Area Number |
01870003
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | University of Tokyo, Faculty of Medicine |
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Principal Investigator |
HAGA Tatsuya University of Tokyo, Faculty of Medicine Professor, 医学部(医), 教授 (30011646)
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| Co-Investigator(Kenkyū-buntansha) |
町田 肇 三菱化成(株), 総合研究所, 主任研究員
NODA Kouji Asahi-Kasei Co. Ltd., Chief Researcher, GS開発室, 室長
ISHIDA Torao Asahi-Kasei Co. Ltd., Institute for Life Sciences, Director, ライフサイエンス総合研究所, 所長
NOGUCHI Hajime Mitsubishi-Kasei Co. Ltd., Chief Researcher
野田 康二 旭化成, 開発室, 室長
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| Project Period (FY) |
1989 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥22,400,000 (Direct Cost: ¥22,400,000)
Fiscal Year 1991: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1990: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1989: ¥13,400,000 (Direct Cost: ¥13,400,000)
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| Keywords | G Protein / Acetylcholine Receptor / Muscarinic Receptor / Protein Purification / Affinity Chromatography / Receptor / GTP-Binding Protein / Baculovirus |
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| Research Abstract |
The purpose of the present project is to develop a general method to purify G protein-linked receptors. The strategy and initial schedule are as follows ; (1) purification of a large quantity of muscarinic receptors (mAChRs) which will be used as a model system, (2) studies of the interaction of mAChrS and G proteins in the reconstitution system, (3) solubilization of the complex of mAChRs and G proteins, (4) isolation of receptors by the use of an affinity chromatography with G proteins or anti-G proteins as ligands and a specific elution of receptors from these gels by agonists of GTP. An introduction of baculovirus-insect cell (Sf9) system and the improvement of the affinity column enablied us to purify mAChRs (m2) in a relatively high quantity and reproducebly. Detailed studies on the interaction of mAChRs (m2) and G proteins (Gi, Go, Gn) in reconstitution system have indicatied that mAChRs from the complex with the granine nucleotide-free G proteins with or without Mg^<2+> but the complex in the presence of Mg^<2+> has higher affinities for agonists and is considered to be an intermediate for the action of mAChR on G proteins. Inhibitory effects of G proteins on the agonist-dependent phosphorylation of muscarinic receptors by mAChR kinase also indicated the formation of the complex. The complex of mAChR and G proteins was found to be solubilized with sodium monolaurate from atrial membranes which had been incubated with G proteins and to be formed in the same detergent solution. We have been attempting to bind mAChRs or mAChR-G protein complex, which were solubilized with sodium monolaurate, to agarose gels with G proteins or anti-G proteins as ligands and to elute them from these gels specifically, but we have not yet found the satisfactory conditions.
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