2005 Fiscal Year Final Research Report Summary
Crystallization of bovine heart mitochondrial respiratory complex I
| Project/Area Number |
16570098
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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 |
Structural biochemistry
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| Research Institution | University of Hyogo |
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Principal Investigator |
SHINZAWA Kyoko (新澤 恭子) University of Hyogo, Graduate School of Life Science, Assistant, 大学院生命理学研究科, 助手 (70206316)
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| Project Period (FY) |
2004 – 2005
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| Keywords | mitochondrial respiratory chain / complex I / membrane protein complex / crystallization |
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| Research Abstract |
Complex I (NADH-ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chains. It catalyzes the transfer of two electrons from NADH to quinine, coupled to the translocation of about four protons across the membrane. The mitochondrial enzyme contain 46 different subunits and is one of the largest known membrane protein complex, the molecular mass of about 1000kDa. To elucidate the mechanism of this big membrane protein complex reactions, the three dimensional structure is indispensable.
For making the crystals of this membrane complex, the native enzyme must be purified from bovine heart mitochondrial membrane. NADH-Q1 oxidoreducutase activities are inhibited by piericidin A or rotenone specifically in the mitochondrial membrane, but the sensitivity to these inhibitors is decreased if some denaturalization occur.
The method for purification of this enzyme were established. Enzymes were solubilized with deoxycholate and DDM and DM, and applied to anionic exchange sepharose chromatography. The new methods needs only two days. The purified enzyme had 2-fold higher activities than reported one, high sensitivities for inhibitors (>93%). The enzyme preparation from different batch contained about 200 molecules of phospholipids, suggested these phospholipids were essential to stabilize this enzyme. The purified protein in DM solution is mixed with PC, then DM was removed by dialysis. Enzymes form 2D lattices, but small area, under some conditions. The crystal-like masses, which exhibit color of this enzyme, were obtained under vapor-diffusion methods.
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Research Products
(2 results)
