白鲢鱼背侧肌焦磷酸酶的纯化及酶学特性

Purification and Characterization of Pyrophosphatase from Dorsal Muscle in Silver Carp(Hypophthalmichthys molitrix)

通过粗分离、50%~80%饱和度硫酸铵分级沉降、DE-52阴离子交换柱层析等步骤,从白鲢鱼背侧肌中分离纯化出焦磷酸酶。通过变性凝胶电泳分析,该酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱中仅有一个分子质量为40 kD的条带。酶学特性研究表明,白鲢鱼背侧肌焦磷酸酶可专一性地水解焦磷酸盐,反应初速率时间范围为0~15 min,最适反应温度为45℃,最适反应pH值为7.5。Mg^(2+)对该酶有明显的激活作用,在Mg^(2+)浓度为5 mmol/L时酶活力最高。5 mmol/L的Ca^(2+)、Zn^(2+)、乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)-Na_2、EDTA-Na_4、KIO_3和1 mmol/L的NaF均对该酶有强烈的抑制作用。该酶水解焦磷酸四钠的最大反应速率为0.051 U/mg,米氏常数为0.54 mmol/L。

Pyrophosphatase（PPase） was extracted and purified from silver carp（Hypophthalmichthys molitrix） dorsal muscle by crude extraction, 50%–80% saturated ammonium sulfate precipitation and DE-52 anion exchange column chromatography. The purified enzyme migrated as a single band with a molecular mass of 40 k D on SDS-polyacrylamide gel electrophoresis（SDS-PAGE）. The enzymatic characterization showed that the purified enzyme could hydrolyze pyrophosphates, and the time range for its initial velocity was 0–15 min. The optimum temperature for the purified PPase was 45 ℃ and the optimum p H was 7.5. Mg^（2＋） was necessary for PPase activation and the activity reached up to the maximum value at Mg^（2＋）concentration of 5 mmol/L. The activity of PPase was strongly inhibited by 5 mmol/L Ca^（2＋）, Zn^（2＋）, EDTA-Na_2, EDTA-Na_4, KIO3 or 1 mmol/L Na F. The kinetic constants vmax and Km of the purified PPase for tetrasodium pyrophosphate as substrate were 0.051 U/mg and 0.54 mmol/L, respectively.