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.
出处
《热带亚热带植物学报》
CAS
CSCD
北大核心
2001年第1期14-18,共5页
Journal of Tropical and Subtropical Botany
基金
国家自然科学基金项目!(39800009)
广东省自然科学基金项目!(970308)
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
Glycolate oxidase isozyme
Spinach
Catalyzed property
Isoelectric point
Molecular weight
Polymeric form
