Analysis of structure and function of flavin enzyme in o_<> ^--generating enzyme of quinea pig phagocytes.
| Project/Area Number |
61571060
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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 | HIGASHI-NIPPON-GAKUEN-UNIVERSITY |
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Principal Investigator |
TAMOTO Koichi Higashi-Nippon-Gakuen university, Faculty of Pharmaceutical Scienes, Assistant Professor, 薬学部, 助教授 (50088861)
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| Co-Investigator(Kenkyū-buntansha) |
KAZUNOBU Miura Hokkaido University, Faculty of Pharmaceutical Sciences, Lecturer, 薬学部, 助手 (70001980)
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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: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1986: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | O_2^--generating enzyme / flavoprotein / NADPH-cytochrome c reductase / macrophages / protein phosphorylation / 蛋白質リン酸化 / スーパーオキサイドアニオン生成酵素 / 抗NADPH-チトクロムP450 reductase抗体 |
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| Research Abstract |
Upon exposure to appropriate stimuli, macrophages(Mphi) and polymorphonuclear leukocytes(PMN) generate superoxide(O_2^-). The O_2^--generating enzyme of these cells is known as a plasma membrane-associated NADPH oxidase which is consisting of several components, such as flavoprotein and cytochrome b_<558>. However, identity of the flavoprotein and a precise mechanism by which the NADPH oxidase is activated are still obscure. We previously observed that enhanced NADPH oxidase activity of the membrane fraction isolated from phorbol-myristate acetate-activated guinea pig PMN was specifically inhibited by rabbit antibody to homologous liver NADPH-cytochrome c reductase (reductase). Based on this result, in this study, we have examined if alterlation of the membrane-bound reductase of phagocytes in the subcellular localization or in the structure occurs before and after the stimulation of guinea pig M<ph> and PMN. The results of this study are summerized as follows; Although the study at the level of electron microscope is still now in progress, an ultracentrifugation study using percoll discontinuous density gradients revealed that subcellular localization of the reductase did not alter before and after stimulation of the cells with phorbol ester,TPA, and the enzyme firmly bound to a plasma membrane. The enzyme was found to have a molecular weight of 80kd when the affinity purified enzyme was analyzed on SDS-PAGE. Hen M<ph> and PMN were stimulated with TPA and fMLP, it was found that the reductase was phosphorylated with time after stimulation, accompanying the activation of NADPH oxidase. In addition, dephosphorylation of the enzyme was attended by decrease in O_2^--generating enzyme activity.
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Report
(2 results)
Research Products
(2 results)
