| Project/Area Number |
61570151
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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 | National Cardiovascular Center Research Institute |
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Principal Investigator |
MIYAKE Yoshihiro Department of Biochemistry, National Cardiovascular Center Research Institute,Head, 生化学部, 部長 (40028353)
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| Co-Investigator(Kenkyū-buntansha) |
高橋 砂織 国立循環器病センター研究所, 生化学部, 研究員 (10142184)
WATANABE Fusao Department of Biochemistry, National Cardiovascular Center Reserach Institute, R, 生化学部, 研究員 (40183719)
MOMOI Kyoko Department of Biochemistry, National Cardiovascular Center Research Institute, R, 生化学部, 研究員 (90174359)
FUKUI Kyoshi Department of biochemistry, National Cardiovascular Center Research Institute, S, 生化学部, 室長 (00175564)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1987: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | LLC-PK1 cells / D-amino acid oxidase / cDNA / site-directed mutagenesis / Prokallikrein / human urine / complete amino acid sepuence / D-アミノ酸オキシダーゼ欠損マウス / LLC-P【K_1】細胞 / 人尿プロカリクレイン / レニン結合蛋白質 / グルコース膜輸送 |
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| Research Abstract |
19Oxidation product of D-propargylglycine from D-amino acid oxidase(DAO) reaction inhibited uptake of (14C) -<Alpha>-methylglucoside into LLC-PK_1 cells,an established cell line from proximal tubules of porcine kidney.The extent of inhibition was 95% of that of normal uptake.The chemical structure of the product could not be detemined owing to unstability after isolation.However,analysis of the material by^1H-NMR spectroscopy suggests that it is 2-amino-4-hydroxy-2,4-pentadienoate-<gamma>-lactone.
2.An apparatus for NMR measurement of ion transport across a monolayer of LLC-PK1 cells on a nitrocellulose filter has been contrived,and Na^+ transport was measured.
3.Complimentary DNAs encoding DAO have been isolated from porcins kidney cDNA library by hybridization with synthetic oligonucleotides and the nucleotide sequences were determined. In vitro DAO synthesizing system has been established using the isolated clone,and in vitro syntesizing system of mutant DAOs also was established afte
… More
r replacement of single nucleotide of the clone by site-directed mutagenesis.As the results,Tyr(228) and His(307),both modified by D-propargylglycine,were shown to be essential for essential for maintaining DAO activity. Kidney homogenates from ddY-strain mice exhibited various DAO activity.It is indicated from southern and northern blot analyses that the difference in DAO activity caused transcriptional control of DAO gene expression.
4.The activation mechanism of human urinary prokallidrein has been investigated using trypsin as a model activator.The rapid appearance of kallikrein activity by the reaction of prokallikrein with trypsin was due to release of the propeptide from prokallikrein,and the slow increase in kallikrein activity after the rapid activation was due to conversion of the single chain kallikrein to two chain form. The complete amino acid sequence has been determined.As the result,again,it has been demonstrated that the rapid activation is due to cleavage of Arb(-1) -Ile(1) bond and the slow increase in kallikrein activity is due to cleavage of Arg(87)-Gln(88) bond.The presence of oligosaccharide binding site at Asn(78), (84),and (141) was indicated from the amino acid sequence.Immunochemical properties of prokallikrein differed from those of kallikrein,although the difference in molecular structure between the two forms of kallikrein is slight. Less
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