| Project/Area Number |
63480505
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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 |
代謝生物化学
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| Research Institution | Keio University |
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Principal Investigator |
ISHIMURA Yuzuru Keio Univ. School of Med. Professor, 医学部・医化学, 教授 (40025599)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAGI Yasumitsu Keio Univ. School of Med. Instructor, 医学部・医化学, 助手 (20212003)
TANAKA Torahiko Keio Univ. School of Med. Instructor, 医学部・医化学, 助手 (90171785)
MAKINO Ryu Keio Univ. School of Med. Assistant Professor, 医学部・医化学, 講師 (40101026)
荻島 正 慶応義塾大学, 医学部, 助手 (70177153)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1989: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1988: ¥5,800,000 (Direct Cost: ¥5,800,000)
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| Keywords | Polymorphonuclear Leukocyte / Superoxide / NADPH oxidoreductase / Cytosolic Factor / Thyroid Follicular Cell / Fatty Acid / ス-パ-オキサイド / 甲状腺濾胞細胞 / 多形核白血球 / 甲状腺細胞 / スーパーオキシドアニオン / O^-_2 / 活性酸素 |
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| Research Abstract |
1. O^-_ generating system in PMNs
O^-_ generating enzyme system in polymorphonuclear leukocytes (PMNS) is called NADPH oxidase. We studied the activation mechanisms of the oxidase in a cell-free preparation from porcine PMNS. To activate the oxidase in vitro, membrane fraction (the enzyme), cytosol fraction and fatty acid were required. The activation was reversible. The activated oxidase was deactivated by removal of fatty acid and reactivated by replenishment of both fatty acid and fresh cytosol fraction. Effective component in cytosol fraction (cytosolic factor, CF) seemed to be consumed or translocated to the membrane fraction during the activation. To clarify further mechanisms of the activation we tried to purify and characterize the CF. By anion exchange chromatography it was fractionated into two essential components. One of them was purified to near homogeneity which gave a main band at 60 kD on SDS polyacrylamide gel electrophoresis. Antibody raised against the 60 kD protein inhibited the activation of the oxidase. Function of the CF is under investigation. Purification of another CF and components of NADPH oxidase is now in progress.
2. O^-_ generating system in thyroid cells
It is widely known that hydrogen peroxide (H_2O_2) is required for thyroid hormone biosynthesis. However, little is known about molecular mechanisms of the H_2O_2 forming oxidase system in thyroid cells. Our new method for discrimination between H_2O_2 and O^-_ revealed that the plasma membrane fraction of the cells produced 02- as the primary metabolite of oxygen by using NADPH as an electron donor. On the other hand, intact thyroid cells released only H_2O_2 into extracellular medium. As superoxide dismutase activity in the cells was more than 10 times higher than that in neutrophils, we concluded that O^-_ was produced by the oxidase initially and then efficiently dismutated to H_2O_2 for the reaction with thyroid peroxidase in physiological conditions.
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