Starling J A, Allen B L, Kaeini M R, Payne D M, Blytt H J, Hofer H W, Harris B G
J Biol Chem. 1982 Apr 10;257(7):3795-800.
A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.
已开发出一种快速高效的方法，用于从寄生蛔虫猪蛔虫的肌肉中纯化磷酸果糖激酶。该方法可在1天内完成，纯化倍数为420倍，产率为60%。通过二维电泳、琼脂糖6B柱色谱和使用尺寸排阻柱的高效液相色谱法，证明该酶是纯的。在十二烷基硫酸钠存在下进行电泳，发现该酶的亚基分子量为95,000。在低离子强度溶液中，天然酶聚集形成的分子种类比兔肌肉磷酸果糖激酶的分子种类分子量更高。在0.2 M硫酸铵存在下，通过高效液相色谱法和琼脂糖6B柱色谱法测定天然酶的最小分子量为398,000。因此，该酶似乎是由相同或近乎相同的亚基组成的四聚体。通过柱和凝胶等电聚焦法，该酶的表观等电点显示为7.3至7.4。氨基酸分析表明，与兔肌肉酶相比，该酶中芳香族残基苯丙氨酸、酪氨酸和色氨酸的数量较少，这与较低的消光系数（E1%280 nm = 6.5）一致。对纯化酶的分析显示，每摩尔酶含有7.4±0.6摩尔磷酸盐。
