| Project/Area Number |
12680655
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Himeji Institute of Technology |
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Principal Investigator |
TSUBAKI Motonai Himeji Institute of Technology Faculty of Science Associate Professor, 大学院・理学研究科, 教授 (80032283)
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| Co-Investigator(Kenkyū-buntansha) |
角田 佳充 大阪大学, 大学院・理学研究科, 助手 (00314360)
大岡 宏造 大阪大学, 大学院・理学研究科, 助手 (30201966)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Vitamin C / ascorbic acid / cytochrome b561 / noradrenaline / catecholamine / chromaffin vesicle / transmembrane electron transfer / heme / RecJ蛋白質 / 高度好熱菌 |
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| Research Abstract |
1. Cytochrome b561
Cytochrome b561 exists in the membranes of neuroendocrine vesicles of central and peripheral nervous systems. It functions as a transmembrane electron conduit ; I. e. receiving one electron equivalent from ascorbate at the intravesicular surface and donating one electron equivalent to monodehydro ascorbate radical at the intravesicular surface. We found previously that cytochrome b561 contained two independent heme centers (gz = 3.12 and gz = 3.69 species) by EPR spectroscopy and heme content analysis.
2. Redox Potential Measurement
Redox potential measurements of native cytochrome b561 in a detergent-solubilized state showed a presence of two redox components with a different midpoint potential (+155 and +62 mV). DEPC treatment of cytochrome b561 in the oxidized state caused a drastic lowering of the lower redox component as low as -30 mV. This observation is consistent with the notion that the DEPC treatment caused an inhibition of electron accepting ability from asco
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rbate by the specific N-carbethoxylations of conserved residues (His88, His161 and Lys85) of cytochorme b561.
3. EPR Spectroscopy
Effect of the DEPC-treatment on the oxidized EPR spectrum of cytochrome b561 was examined. However, there was no appreciable difference between the native and the DEPC-treated samples. Upon addition of ascorbate to the native sample, both heme centers could be fully reduced. However, for the DEPC-treated sample, only the gz = 3.12 signal disappeared. The gz = 3.69 signal remained in full intensity. This result suggests that the gz = 3.69 species has a role for the electron accepting from ascorbate at the extravesicular site and is susceptible to the DEPC-modification.
4. Transmembrane Electron Transfer Supported by Cytochrome b561
Purified cytochrome b561 was successfully reconstituted into ascorbate-loaded vesicle membranes in an inside-out orientation. Cytochrome b561 could donate electron equivalent to extravesicular cytochrome c via transmembrane elecron transfer. Further, cytochrome b561 could support the monooxygenase activity of extravesicular dopamine b-hydroxylase in the absence of any redox mediator. Addition of ferricyanide as a redox mediator enhance the extravesicular monooxygenase acitivity. Less
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