| Project/Area Number |
03671048
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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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 |
NEGISHI Manabu Kyoto University Assistant Professor Faculty of Pharmaceutical Sciences, 薬学部, 助手 (60201696)
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| Co-Investigator(Kenkyū-buntansha) |
ICHIKAWA Atsushi Kyoto University Professor Faculty of Pharmaceutical Sciences, 薬学部, 教授 (10025695)
YATSUNAMI Kimio Kyoto University Assistant Professor Faculty of Pharmaceutical Sciences (30191141)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Prostacyclin / Adenylate cyclase / Cloning / Receptor / Prostaglandin E2 / Mast cell / Photoaffinity / cAMP / トロンボキサンA_2 / ホモロジースクリーニング / PCR法 / フォトアフィニティ- / 癌化肥満細胞 |
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| Research Abstract |
The prostacyclin (PGI_2) receptor of mouse mastocytoma P-815 cells was characterized by photoaffinity labeling with the stable PGI_2 analogue [15-^3H_1]-19-(3-azidophenyl)-20-norisocarbacyclin ([^3H]APNIC) used as a potential photoaffinity probe for the receptor. [^3H]APNIC bound to the mastocytoma membrane with high affinity and in a saturable manner. Scatchard plot analysis indicated a single binding site with a Kd of 4.7 nM and a Bmax of 0.58 pmol/mg protein. The binding of [^3H]APNIC was dose dependently inhibited by APNIC and iloprost, another stable PGI_2 agonist, and to a much lessor extent by PGE_2. The binding of the radioligand showed sensitivity to the guanine nucleotide guanosins 5'-OMICRON-(3-thiotriphosphate) (GTPgammaS). Photolysis of [^3H]APNIC-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 43 kDa. Photolabeling was inhibited by PGI_2 agonists and other prostaglandins with specificity for the PGI_2 receptor and was modulated by GTPgammaS. A protein of approximately 45 kDa was also labeled by [^3H]APNIC in the membrane of porcine platelets, membranes that are known to be abundant in PGI_2 receptors. These results demonstrated that [^3H]APNIC specifically labels a protein that may represent the PGI_2 receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PGI_2 receptor.
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