| Project/Area Number |
12680624
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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 |
Functional biochemistry
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
SAGAMI Ikuko Tohoku University, Institute of Multidisciplinary Research for Advanced Materials, Lecturer, 多元物質科学研究所, 講師 (10143033)
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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 |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | nitric-oxide synthase / calmodulin / autoinhibitory domain / electron transfer mechanism / heme / caveoline / skeletal muscle cells / mutagenesis / ヘム還元 / カルモデュリン / 二量体形成 |
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| Research Abstract |
In neuronal nitric-oxide synthase (nNOS), calmodulin (CaM) binding is thought to trigger electron transfer from the reductase domain to the heme domain, which is essential for O2 activation and NO formation. To elucidate the electron-transfer mechanism, we characterized a series of heterodimers consisting of one full-length nNOS subunit and one oxygenase-domain subunit. The results support an inter-subunit electron-transfer mechanism for the wild-type nNOS, in that electrons for catalysis transfer in a Ca^<2+>/CaM dependent way from the reductase domain of one subunit to the heme of the other subunit, as proposed for iNOS. This suggests that the two different isoforms form similar dimeric complexes. In a series of heterodimers containing a Ca^<2+>/CaM-insensitive mutant (delta40), electrons transferred from the reductase domain to both hemes in a Ca^<2+>/CaM independent way. Thus, in the delta40 mutant electron transfer from the reductase domains to the heme domains can occur via both inter-subunit and intra-subunit mechanisms. However, NO formation activity was exclusively linked to inter-subunit electron transfer and was observed only in the presence of Ca^<2+>/CaM. This suggests that the mechanism of activation of nNOS by CaM is not solely dependent on the activation of electron transfer to the nNOS hemes, but may involve additional structural factors linked to the catalytic action of the heme domain.
Next we constructed an expression system of nNOSμ in E. coli cell. nNOSμ is an isoform of nNOS which specifically expressed in skeletal muscle cells and contains extra-33 amino acids in the autoinhibitory domain of nNOS wild-type. We succeeded to get enough amount of purified nNOSμ protein to characterize. Now we are undergoing to analyze the regulation of NO formation activity of nNOSμ by CaM and caveoline.
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