Purification of Sulphite-Cytochrome c Reductase of Thiobacillus novellus and Reconstitution of Its Sulphite Oxidase System with the Purified Constituents

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Sulphite-cytochrome c reductase (sulphite: ferricytochrome c oxidoreductase, EC 1.8.2.1) derived from Thiobacillus novellus was purified by chromatography on a DEAE-cellulose column and by gel filtration with a Sephadex G-100 column. Although the reductase thus purified moved as a single band both in gel filtration and in isoelectric focusing it was always split into two bands by polyacrylamide gel electrophoresis; the one had the enzymatic activity and showed absorption spectrum of cytochrome, while the other had no activity and was colourless, in contrast with the results reported by Charles and Suzuki [(1966) Biochim. Biophys. Acta 128: 522]. The enzymatic properties of the purified reductase were almost the same as those of the enzyme obtained by Charles and Suzuki. Cytochrome c-551 free of the reductase activity was obtained. Its molecular weight was determined to be 23,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The cytochrome seemed to exist in the organism as a complex with the reductase or a subunit of the enzyme. In the state of the complex with the enzyme, the cytochrome was reduced very quickly on addition of sulphite, while the cytochrome free of the reductase activity was hardly reduced by the enzyme with sulphite. A sulphite oxidase system was reconstituted with the reductase, cytochrome c-550 and cytochrome oxidase high]y purified from the bacterium.
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