| Project/Area Number |
62570125
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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 |
Pathological medical chemistry
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| Research Institution | University of Tokyo |
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Principal Investigator |
WATANABE Tsuyoshi University of Tokyo Hospital, 医学部(病), 助手 (80158641)
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| Co-Investigator(Kenkyū-buntansha) |
HORIE Yukio University of Tokyo Hospital, 医学部(病), ASSISTANT (40211552)
KINOSHITA Makoto University of Tokyo Hospital, 医学部(病), ASSISTANT (70186295)
TERAMOTO Tamio University of Tokyo Hospital, 医学部(病), ASSOCIATE (20133077)
SHIMIZU Takao University of Tokyo, Department of Natrition and Physiological Chemistry, Faculty of Medicine, ASSISTANT (80127092)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1988: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1987: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Prostaglandin / Kidney / 受容体 |
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| Research Abstract |
In order to understand the molecular mechanism of prostaglandin(PG)E_2 action in the renal tubular cells,we attempted to purify canine renal medullary PGE receptor. PGE_2 binding activity could be effectively solubilized in an active form from renal medullary membranes with digitonin. This solubilized binding activity could be partially,but significantly , purified with a sequential use of gel filtration(HPLC),wheat germ lectin,ion exchange(HPLC) and isoelectric focusing columns. The final purified sample had not retained sensitivity to GTP or its analogues,which the starting solubilized materials had,suggesting that the final preparation had lost association with a GTP-binding protein. We could perform reconstitution experiments of the final preparation and purified GTP-binding protein. We tested the possibility of PGE_2 affinity chromatography for further purification using a proplyamido-derivative of PGE_2. However,the derivative had not the binding activity to solubilized binding protein,neglecting that possibility. We also tried to obtain a monoclonal antibody against this PGE receptor using the partially purified preparation as a immunogen. We screened more than three thousand well using immunoprecipitaion methods with staph.A ,but so far could get no positive well. In this context,we are now trying to make the purification scale up and get more effective purification for immunization of mice. On the other hand,we are trying to apply the xenopus oocytes expression system for cloning cDNA encoding renal medullary PGE receptor and have gotten some positive results.
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