菠菜乙醇酸氧化酶同工酶GO Ⅰ的纯化和特性

PURIFICATION AND PROPERTIES OF GLYCOLATE OXIDASE ISOZYME GO I FROM SPINACH LEAVES
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    摘要:

    从菠菜绿叶中获得SDS-PAGE为40 000±2 000Mr单带的乙醇酸氧化酶同工酶GO I。其GO比活为8.4 U min-1mg-1 protein,经ND-PAGE后显示Mr为470 000。用IEF测得其等电位为7.4。用氧电极法证实该GO I能同时催化乙醇酸和乙醛酸的氧化,制备GO I的抗体并对菠菜绿叶粗蛋白作免疫双扩散,有免疫沉淀线,经蛋白A-Sepharose CL-4B柱吸附法和抗原亲和吸附法,得到单特异性GO I抗体,用该GO I抗体对菠 菜绿叶粗蛋白作SDS-PAGE Western blot后只有40 000±2 000-一条染色带,作4%-20%PAGE Western lot后有多条 色带,Mr分别大约为470 000,280 000和80 000,表明菠菜绿叶中的GO I有多个聚合态。

    Abstract:

    By using ammonium sulfate fractionation, Sephadex G-25, DEAE-Cellulose, and Sepharose-6B chromatographies, glycolate oxidase isozyme GO I was purified from spinach green leaves which showed only one band with molecular weight (Mr) of 40 000± 2 000 in SDS-PAGE. The specific activity of purified GO I was 8.4 U min-1mg-1 protein. GO I showed its Mr of 470 000 in 4%-20% ND-PAGE and could catalyze both oxidations of glycolate and glyoxylate simultaneously. The isoelectric point (PI) of GO I was about PH 7.4. There was a immunity precipitate line determined by immunity double-difusion reaction between the antibody raised against GO I and crude protein from spinach green leaves.The specitic G0 I antibody was purified by protein A-Sepharose CL-4B column and specific antigen affinity elution.Only the 40 000± 2 000 band could immunity cross-react with Go I antibody,and different Go I polvmers of 470 000,280 000,and 80000 were found when crude protein were conduced to SDS-PAGE Ⅵ stern blot or ND-PAGE Western blot using this speciftc GO I antibody.

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徐杰, 吴燕燕.菠菜乙醇酸氧化酶同工酶GO Ⅰ的纯化和特性[J].热带亚热带植物学报,2001,9(1):14~18

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