作　　者： ; ;

机构地区： 华南师范大学生命科学学院生物技术研究所

出　　处： 《热带亚热带植物学报》 2001年第1期14-18,共5页

摘　　要： 从菠菜绿叶中获得ＳＤＳ－ＰＡＧＥ为４０ ０００±２０００Ｍｒ单带的乙醇酸氧化酶同工酶ＧＯⅠ。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ＧＯⅠ能同时催化乙醇酸和乙醛酸的氧化。制备ＧＯⅠ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ＧＯⅠ抗体。用该ＧＯⅠ抗体对菠菜绿叶粗蛋白作ＳＤＳ－ＰＡＧＥ Ｗｅｓｔｅｒｎ ｂｌｏｔ后只有４００００±２０００－条染色带；作 ４％－ ２０％ＮＤ－ＰＡＧＥ Ｗｅｓｔｅｒｎ ｂｌｏｔ后有多条染色带， Ｍｒ分别大约为 ４７０ ０００、 ２８０ ０００和８０ ０００，表明菠菜绿叶中的 ＧＯＩ有多个聚合态。 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 40000± 2000 in SDS-PAGE. The specific activity of purified GO I was 8.4 U min-1mg-1 protein. GO I showed its Mr of 470000 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-diffusion reaction between the antibody raised against GO I and crude protein from spinach green leaves. The specific GO I antibody was purified by protein A-Sepharose CL-4B column and specific antigen affinity elution. Only the 40000 ±2000 band could immunity cross-react with GO I antibody, and different GO I polymers of 470000, 280000, and 80000 were found when crude protein were conduced to SDS-PAGE Western blot or ND-PAGE Western blot using this specific GO I antibody.

关 键 词： 乙醇酸氧化酶同工酶 菠菜 催化特性 等电点 分子量 聚合态 纯化 植物

领　　域： [生物学]