| Project/Area Number |
62440028
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Kyushu University |
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Principal Investigator |
MINAKAMI Shigeki Kyushu University, Faculty of Medicine, Professor, 医学部, 教授 (90037325)
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| Co-Investigator(Kenkyū-buntansha) |
NANRI Hiroki Kyushu University, Faculty of Medicine, Research Associate, 医学部, 助手 (80150415)
TAKESHIGE Koichiro Kyushu University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (10037450)
住本 英樹 九州大学, 医学部, 助手 (30179303)
日野 幸伸 九州大学, 医学部, 講師 (40112338)
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| Project Period (FY) |
1987 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 1990: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1988: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥9,600,000 (Direct Cost: ¥9,600,000)
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| Keywords | Neutrophils / NADPH oxidase / Superoxide / Plasma membrane / Phospholipid / Cytochrome b_<558> / 活性酸素 / グアニンヌクレオチド / アラキドン酸 / NADPHオキシダーゼ / チトクロム_558 / 慢性肉芽腫症 |
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| Research Abstract |
The enzymatic activity responsible for the respiratory burst is that of the membranebound NADPH oxidase, which reduces molecular oxygen to superoxide. This enzyme has been postulated to be a multicomponent electron transport chain consisting of a flavo protein and a cytochrome b_<558> We have studied the roles of the cytochrome and cytocolic factors in the activation of the oxidase in a cell-free system.
1. Analyses of the neutrophil cytosolic factors
The potency of the cytosol fraction of pig neutrophils in the activation of the superoxide production could be reconstituted dose-dependently by mixing two protein components with relative molecular masses of 300 kDa and 50 kDa, which were separated by gel filtration. The cytosol fraction was analyzed by chromatography on 2', 5'-ADP agarose and two components responsible for the GTP-dependent and independent activation were separated, indecating that at least two pathways are available for the activation of superoxide production in the cell-free system. We found that the NADPH binding component of the oxidase is present in the 300KDa component by the studies using 2', 3'-dialdehyde NADPH.
2. Cytochrome b_<558> as a component of the NADPH oxidase
The membrane factors were effectively extracted by 0.75% octyl glucoside and analyzed by chromotography on wheat-germ-agglutinin-agarose. The results show that cytochrome b_<558> is probably a membrane component of the oxidase aud is activation in the cell-free system requires the reconstitution with phospholipids. A cDNA clone for the mouse homolog of the small subunit of human cytochrome b_<558> was isolated. The predicted amino acid sequence had a high degree of homology (87%) with the human protein. The mRNA expressed in the mouse kidney, spleen and small intestine as well as phagocytes, suggesting another function of the small subunit.
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