| Project/Area Number |
01571195
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
Biological pharmacy
|
|---|
| Research Institution | Chiba University |
|---|
Principal Investigator |
UNEMOTO Tsutomu Chiba University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (30089601)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Maki Chiba University, Faculty of Pharmaceutical Sciences, Associate Professor, 薬学部, 助教授 (50092086)
|
|---|
| Project Period (FY) |
1989 – 1990
|
| Project Status |
Completed (Fiscal Year 1990)
|
| Budget Amount *help |
¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1990: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1989: ¥800,000 (Direct Cost: ¥800,000)
|
|---|
| Keywords | Menadione / NADH-quinone reductase / Induction / DT-Diaphorase / Quinone toxicity / E. coli / Oxygen stress / Quinone metabolism / 毒性 / NADH-quinone reductase / DT-diaphorase / キノン / 活性酵素 / 一電子還元 / 二電子還元 |
|---|
| Research Abstract |
It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1, 4-naphthoquinone), an FMN-dependent NADH-quinonereducase increases more than 20-fold in the cytoplasmic fraction. The menadion-induced quinone reductase was isolated from the cyto plasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 muM FMN or 16.5 muM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADA and menadione were estimated to be 132 muM and 2.0 muM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 muM. The electron acceptor specificity of this enzyme was very similar to that of NAD (P) H : (quinone acceptor) oxidoreductase (EC1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, The induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
|
