萌发木豆种子中酸性磷酸酯酶的纯化和动力学特性(英文)  

Purification and kinetic characteristics of an acid phosphatase isoform from germinating pigeonpea seed

作　　者：董登峰[1] 李杨瑞 杨杰[2] 王永峰[2] 

机构地区：[1]广西作物遗传改良和生物技术重点实验室,南宁530007 [2]广西大学农学院,南宁530004

出　　处：《广西植物》2008年第3期390-394,共5页Guihaia

基　　金：Supported by Postdoctoral Research Foundation of Guangxi Academy of Agriculture Sciences;Natural Science Foundation of College of Agricultural,Guangxi University

摘　　要：以对硝基苯磷酸为底物检测酶活性,通过20%～60%硫酸铵分部、DEAE-葡聚糖A25、羟基磷灰石、伴刀豆球蛋白-琼脂糖4B柱层析,从木豆萌发的种子中纯化到一个同工酶APaseⅡ,酶最终纯化倍数为247倍,比活力达51.8U/mg蛋白。非变性PAGE和SDS-PAGE表明所纯化的酶已经达到电泳纯,是一个分子量为33.1kDa的单体蛋白。APII的最适pH为5.0,最适温度为35℃,在pH3.5～7以及55℃以下稳定。该酶对焦磷酸有最大活性,受K+和Mg2+激活,受Fe2+,Mn2+,Mo7O246-,F-及酒石酸、苹果酸、异柠檬酸、草酸、柠檬酸、乙醇酸、乙醛酸和抗坏血酸等有机酸抑制。Using p-nitrophenolphosphate（pNPP）as substrate, one isoform of acid phosphatase from germinating pi- geonpea seed, encoded as APase Ⅱ, was purified to 247 folds and the specific activity 51.8 U/mg protein through ammonium sulfate fractionation and three sequential DEAE-Sephadex A 25, Hydroxyapatite and Concanavalin A-Sepharose 4B column chromatography. The purified APⅡ was demonstrated by Native- and SDS-PAGE to be electro- phoretic homogeneity and as a 33 kDa monomer. APase Ⅱ exhibited optimal pH at 5.0 and optimal temperature at 35 ℃ and strong stabilization at the pH ranging from 3.5 to 7.0 and at temperature below 55 ℃ APase Ⅱ showed a highest specificity against pyrophosphate, and was activated by K^＋ and Mg^2＋ , while inhibited by Fe^2＋ , Mn^2＋ , Mo7O246^-, F- as well as by organic acids including as tartrate, malate, isocitrate, oxalate, citrate, glycolate, glyoxylate and ascorbate.

关 键 词：木豆 酸性磷酸酯酶 纯化 动力学特性 

分 类 号：Q556.1[生物学—生物化学]