| Project/Area Number |
12671819
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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 basic dentistry
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| Research Institution | Matsumoto Dental University |
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Principal Investigator |
HIRAOKA B.yukihiro Matsumoto Dental University, Dept. of Oral Biochemistry, Associate Professor, 歯学部, 講師 (20097512)
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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 |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | Reactive oxygen / SOD / Superoxyde dismutase / Metallo enzyme / Oxidoreductase / Porphyromanas gingivalis / Metal-specific activity / Site-directed mutagenesis / Porphyromonas gingivalis |
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| Research Abstract |
Active site of Mn and Fe-superoxide dismutases (SODs) have very similar primary and tertiary structure, however, the expression of enzymatic activity of most SODs have strict specificity for the metal at the active site. While P. gingivalies(P.g.) SOD shows exceptionally activity with either Mn or Fe incorporated into the same active site. We have prepared the mutant of the P.g.-SOD with conversions of above amino acid residues to Mn- or Fe-type, in order to clarify contribution of these residues to the metal-specific acitivty of P.g.-SOD.
[RESULTS] We made the Mn- and Fe-type mutation, by site-directed mutagenesis and expressed as a soluble fusion protein coupled to maltose binding protein (MBP) using the expression vector pMAL-c2. After an affinity purification and cleavage MBP by trypsin, the enzyme was purified by anion exchange chromatography with yield of 15-20 mg/l of culture. Wild, the Mn- and Fe-type mutant enzymes were reconstituted with Mn or Fe by using an acid-guanidine-HCl method and purified to homogeneous by a hyroxyapatite-HPLC. The ratio of the specific activity of Mn-/Fe-reconstituted SOD was about 1.5 in the native SOD, to 2.4 in the Mn-type mutant SOD and to 13.4 in the Fe-type mutant SOD. In next study, molecular evolution maybe useful to search for unknown amino acid residues which contribute to the metal-specific activity of Mn-, Fe- and cambialistic SODs.
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