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

摘要:

从菠菜绿叶中获得SDS－PAGE为40 000±2 000M_r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M_r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，M_r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 (M_r) 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 M_r 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.