Isolation and Partial Characterization of Homogeneous Ferredoxin-sulfite Reductase from a Red Alga, Porphyra yezoensis(Biological Chemistry)
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概要
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Ferredoxin-dependent sulfite reductase (Fd-SiR) (EC 1.8.7.1) was purified about 1136-fold, with a yield of 11%, from fresh thalli of Porphyra yezoensis by a procedure involving ammonium sulfate precipitation, DEAE-cellulose chromatography, Butyl-Toyopearl chromatography, Sephadex G-100 gel filtration and ferredoxin-Sepharose affinity chromatography. The purified enzyme was apparently homogeneous, as judged on polyacrylamide disc gel electrophoresis, with a specific activity of 100 units/mg of protein. The molecular weight of the enzyme was estimated to be 70 kilodaltons by gel filtration. On subunit analysis by SDS-PAGE, a single band corresponding to molecular weight of 65 kilodaltons appeared. The purified enzyme (Fd-SiR) showed 5-times higher ferredoxin-dependent activity than methyl viologen-linked activity. In the oxidized form, the enzyme exhibited absorption maxima at 278,390 (Soret band), 586 (α band) and 714 (CT band) nm, indicating that siroheme is involved in the catalysis of sulfite reduction. The absorbance ratios, A_<390>: A_<278> and A_<586> : A_<390>, were 0.32 and 0.31, respectively. A plot of the substrate (sulfite) and electron donor (ferredoxin) concentrations versus enzymatic (Fd-SiR) activity yielded sigmoidal curves, giving Hill coefficients (ii) of 2.3 (for sulfite) and 2.7 (for ferredoxin), respectively. Antibody against the isolated enzyme was raised in rabbits. Analysis of the antiserum by immunodiffusion suggested that it was specific against isolated Fd-SiR. Using the antiserum, dot immunoblotting was performed to determine the immunological similarity of Fd-SiRs from Porphyra yezoensis, Spirulina platensis, Brassica chinensis and Spinacia oleracea. The tests revealed that the four forms of assimilatory Fd-SiR have antigenic determinants in common.
- 社団法人日本農芸化学会の論文
- 1989-06-23
著者
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TAMURA Goro
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University
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Tamura Goro
Department Of Agricultural Chemistry Faclty Of Horticulture Chiba University
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KOGUCHI Osamu
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University
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Koguchi Osamu
Department Of Agricultural Chemistry Faclty Of Horticulture Chiba University
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