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 phos-phatase isoform from germinating pigeonpea seed

关键词:  木豆  酸性磷酸酯酶  纯化  动力学特性

Key words:  pigeonpea  acid phosphatase  purification  kinetic characteristics

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APase Ⅱ,酶最终纯化倍数为247倍,比活力达51.8 U/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。

Abstract:

Using p-nitrophenolphosphate(pNPP)as substrate,one isoform of acid phosphatase from germinating pigeonpea 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 electrophoretic 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 Mg²⁺,while inhibited by Fe²⁺,Mn²⁺,Mo₇O₂₄₆^-,F- as well as by organic acids including as tartrate,malate,isocitrate,oxalate,citrate,glycolate,glyoxylate and ascorbate.