| Project/Area Number |
09650871
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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 |
生物・生体工学
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| Research Institution | Yamanashi University |
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Principal Investigator |
AMANO Yoshifumi Yamanashi Univ.Fac.Eng., Professor, 工学部, 教授 (70020401)
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| Co-Investigator(Kenkyū-buntansha) |
KUROSAWA Hiroshi Yamanashi Univ.Fac.Eng., Assistant Professor, 工学部, 助教授 (10225295)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Thiobacillus thiooxidans / Axid Tolerance / Cytochrome c oxidase / Membrane-bound cytochrome c / シトクロムcオキシダーゼ / シトクロム c オキシダーゼ |
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| Research Abstract |
We studied isolations and properties of cytochrome c oxidase and membrane-bound cytochrome C to elucidate acid tolerant function of Thiobactillus thiooxidans grown under acid and extreme environment.
1. Purification of cytochrome c oxidase : Cell suspension of T.thiooxidans was disrupted and the homogenate was centrifuged to prepare the cell-free extracts. The membrane fraction was prepared by centrifuging the cell-free extracts. The activity of cytochrome C oxidase was estimated by measuring the decrease rate of the absorbance at 550nm due to the oxidation of reduced cytochrome c. Enzymatic properties of the membrane fractions were studied. Optimum pH and temperature of the enzyme reaction was 5.0 and 350C, respectively. The enzyme activity of the membrane fractions was stable at pH 4.0. The enzyme was solubilized with Triton X-100 from the membrane fractions. The Isoelectric point of the enzyme in the solubilized fraction was found to be 5.5 by detection of the indophenol blue band on
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the isoelectric focusing gel. The solubulized fraction was subjected to CM-cellulose and Sephacryl S-200 colums In the presence of Triton X- 100 at pH 4.0. The purified enzyme fraction had two protein bands by native PAGE.Therefore, the enzyme preparation was not homogeneous.
2. Purification of membrane-bound cytochrome C : The fraction solubilized with Triton X- 100 from membrane fraction was subjected to isoelectric focusing. By staining the heme protein bands on the gel, it was found that the solubilized fraction was composed of three different cytochrome c proteins, with isoelectric points of 5.3, 5.8 and 8.2, respectively. The acidic proteins (p1 5.3 and p1 5.8) were subjected to DEAE-Sepharose column in the presence of Triton X- 100 at pH 7.0. However, the protein exihibiting an absorption spectrum of cytochrome c was not observed in the eluted fractions. The basic protein (p1 8.2) was chromatographed with CM-cellulose and hydroxyapatite colums in the presence of Triton X- 100 at pH 7.0. A protein exihibiting the absorption spectrum of cytochrome c was observed in the eluted fractions. The cytochrome c fraction was homogeneous on SDS-PAGE, The molecular weight of the membrane-bound cytochrome c was estimated to be 27kD. Less
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