猪背最长肌焦磷酸酶的分离纯化与酶学特性

通过离心、50%～70%饱和硫酸铵沉淀、DEAE-52离子交换柱层析,从猪背最长肌中分离纯化出焦磷酸酶(PPase)。变性聚丙烯酰胺凝胶电泳图谱显示,PPase分子质量约72kD。焦磷酸酶酶学特性研究表明,最适反应温度和pH值分别为50℃和7.5。Mg2+是PPase的激活剂,在浓度4.75mmol/L时,酶活力最强。但Na+和K+都能抑制酶的活力,且Na+的抑制效果强于K+。PPase水解焦磷酸钠(TSPP)的动力学参数Vmax为0.086μmol/(L.min)),Km为0.36mmol/L。

Purification and Characterization of Pyrophosphatase from Pork longissimus dorsi Muscle

Pyrophosphatase(PPase) from pork longissimus dorsi muscle was purified by ultracentrifugation,50%-70% saturated ammonium sulfate fractionation,DEAE-52 anion-exchange chromatography.The purified enzyme with molecular mass of 72 kD ran as a single band on SDS-polyacrylamide gel.The optimum pH and temperature for the isolated PPase was 7.5 and 50 ℃,respectively.Mg2+ was necessary for PPase and the activity reached maximum at a concentration of 4.75 mmol/L.Na+ and K+ inhibited the enzyme activity and the inhibitory effect of Na+ was stronger than K+.The kinetic constant Km and Vmax using TSPP as substrate were determined as 0.36 mmol/L and 0.086μmol/(L·min),respectively.