Purification and Characterization of Polyphenol Oxidase from Callus Cultures of Portulaca grandiflora

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Three isoforms of polyphenol oxidase (I-a, I-b and II) were purified from betacyanin-producing callus cultures of Portulaca grandiflora, by ammonium sulfate precipitation followed by successive column chromatographies of DEAE cellulose, Sephacryl S-200, hydroxyapatite and Mono-Q. The molecular masses of the monomeric polyphenol oxidase I-a and I-b were calculated to be 66 and 65 kDa, respectively, when analyzed by SDS-PAGE. Polyphenol oxidase II appeared to be a heterodimer of two subunits with molecular masses of 25 and 27 kDa. The optimal pH for polyphenol oxidase activity of I-a and I-b was 5.0, and for II was 6.0. These enzymes were markedly inhibited by sulfhydryl binding reagents, ascorbate, kojic acid, copper-chelating reagents and Fe2+. L-DOPA (L-3, 4-dihydroxyphenylalanin) was a good substrate for these enzymes, although no activity with tyrosine and hydroquinone was observed. The apparent Km values of polyphenol oxidase I-a, I-b and II for DOPA were 2.0, 2.2 and 3.5mM, respectively. No monophenol oxidase (tyrosine hydroxylation) activity by these enzymes was observed.
日本植物細胞分子生物学会の論文
2002-06-01