Li Z, Phillips N F
Department of Medicine and Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4983, USA.
Protein Expr Purif. 1995 Jun;6(3):319-28. doi: 10.1006/prep.1995.1042.
The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) from Giardia lamblia has been purified to homogeneity using two methods. The purified enzyme is shown to be homogenous by its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single peak on reverse-phase HPLC. The purified enzyme is a monomeric protein of Mr 64 kDa as determined by SDS-PAGE and native PAGE. The enzyme fractionated as a 67-kDa protein by HPLC gel filtration. The pH optima in either the glycolytic or the gluconeogenic direction is near neutral, which is unlike any of the protozoan or bacterial enzymes. The enzyme displayed typical Michaelis-Menten kinetics. The Km values for pyrophosphate and fructose 6-phosphate were 0.039 +/- 0.005 and 0.25 +/- 0.0196 mM, respectively. The initial velocity for the reverse reaction was also found to be hyperbolic. Different nucleoside triphosphates, including ATP, could not substitute for pyrophosphate as the phosphoryl donor. Similar to the pyrophosphate-dependent enzyme from other anaerobic protozoans and bacteria, the Giardial enzyme was not activated by fructose 2,6-bisphosphate, although this metabolite is a competitive inhibitor with respect to fructose 1,6-bisphosphate in the reverse reaction. The pH optima and the molecular weight properties of the Giardial enzyme are different from those of the two classes of the pyrophosphate-dependent phosphofructokinases.
利用两种方法已将来自蓝氏贾第鞭毛虫的焦磷酸依赖性磷酸果糖激酶（EC 2.7.1.90）纯化至同质。纯化后的酶在十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）上以单一条带迁移，在反相高效液相色谱（HPLC）上呈现单峰，表明其具有同质性。通过SDS - PAGE和天然PAGE测定，纯化后的酶是一种分子量为64 kDa的单体蛋白。通过HPLC凝胶过滤，该酶分级分离为一种67 kDa的蛋白。在糖酵解或糖异生方向上，其最适pH接近中性，这与任何原生动物或细菌的酶都不同。该酶表现出典型的米氏动力学。焦磷酸和6 - 磷酸果糖的Km值分别为0.039±0.005和0.25±0.0196 mM。还发现逆反应的初始速度也是双曲线型的。包括ATP在内的不同核苷三磷酸不能替代焦磷酸作为磷酰基供体。与来自其他厌氧原生动物和细菌的焦磷酸依赖性酶类似，蓝氏贾第鞭毛虫的酶不受2,6 - 二磷酸果糖的激活，尽管这种代谢物在逆反应中是1,6 - 二磷酸果糖的竞争性抑制剂。蓝氏贾第鞭毛虫酶的最适pH和分子量特性与两类焦磷酸依赖性磷酸果糖激酶不同。
