萌发木豆种子中酸性磷酸酯酶的纯化和动力学特性

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。

Purification and kinetic characteristics of an acid phos- phatase isoform from germinating pigeonpea seed

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/rag protein through am- monium sulfate fractionation and three sequential DEAE-Sephadex A 25, Hydroxyapatite and Concanavalin A-Sepha- rose 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 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.