1986 Fiscal Year Final Research Report Summary
Phagocytosis-specific NADPH-oxidase and its Induction by Phagocytosis
| Project/Area Number |
60480138
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
MINAKAMI Shigeki Kyushu Univ., Fac. Med., Professor, 医学部, 教授 (90037325)
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| Co-Investigator(Kenkyū-buntansha) |
SUMIMOTO Hideki Kyushu Univ., Fac. Med., Assistant, 医学部, 助手 (30179303)
HINO Yukinobu Kyushu Univ., Fac. Med., Assistant, 医学部, 助手 (40112338)
TAKESHIGE Koichiro Kyushu Univ., Fac. Med., Assistant professor, 医学部, 助教授 (10037450)
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| Project Period (FY) |
1985 – 1986
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| Keywords | Phagocytosis / Intracellular pH / HADPH-oxidase / NADPH-analog / Superoxide / Cytochrome / Ubiquinone / モノクローン抗体 |
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| Research Abstract |
1. Intracellular pH: The changes of intracelleular pH of human neutrophils treated by chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) and other stimulatnts were analyzed by a fluorescent probe 9-aminoacridine. The initial intracellular acidification and subsequent alcalization were dependent on the extracellular <Ca^(2+)> concentrations, and a calcium ionophore A23187 showed similar changes. The intracellular pH changes due to the peptide was inhibited by pertussis toxin indicating a possibile involvement of a GTP-binding protein. Involvement of the intracellular free <Ca^(2+)> and <Ca^(2+)> , phospholipid-dependent protein kinase on the induction of the phagocytic NADPH oxidase were shown in various phagocyte systems.
2. Functional analysis of NADPH oxidase components: The NADPH oxidase was considered to contain a flavoprotein, ubiquinone and a b-type cytochrome. Functional involvement of ubiquinone has been suggested by an observation that ubiquinone can be reduced by the oxidase in the presence of superoxide dismutase (SOD). We analyzed the reaction and showed that ubiquinone is reduced by superoxide so rapidly that SOD at usual condition could not inhibit the reaction. Thus the functional involvement of ubiquinone in the oxidase became questionable.
3. Purification of the NADPH oxidase: Various chromatographic techniques were applied on the purification of the enzyme, and two specific labeling methods of the enzyme protein(s),by a monoclonal antibody against the b-type cytochrome and by dialdehyde derivative of NADPH, were develped. The localization of the cytochrome in neutrophils of healthy individuals were shown by immuno-electron microscopy but the cytochrome was absent in neutrophils of chronic granulomatous disease patients while the covalently bound NADPH analog in neutrophil phagosomes was not deficient in the patients.
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Research Products
(41 results)
