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大白菜葡萄糖磷酸变位酶同工酶的纯化及其分子性质

DOI: 10.3321/j.issn:1000-7091.1999.01.027, PP. 134-140

Keywords: 葡萄糖磷酸变位酶,同工酶,米氏常数,耐热性,柱层析,大白菜

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Abstract:

首先对影响葡萄糖磷酸变位酶(PGM)稳定性的因素进行了比较研究,结果发现,在所有被检测的因子中,磷酸根对酶的保护作用最强,而Tris-盐酸则效果极差。在磷酸缓冲液中添加二硫苏糖醇、PMSF、葡萄糖和镁离子等可能的保持试剂未能进一步提高酶的稳定性。另外,pH对PGM酶的稳定性有较大影响,酸性pH不利于酶活性的保持,在pH8.0时酶最为稳定。因此,本实验对PGM同工酶的纯化均在pH8.0的磷酸缓冲液中进行。依次经过抽提、分步硫酸铵沉淀、SephadexG75柱层析以及二乙氨乙基纤维素柱高效液相色谱(DEAE-HPLC)等分离步骤,我们纯化了大白菜品种“白阳”黄化种苗中的3个葡萄糖磷酸变位酶(PGM)同工酶,并分别测定了同耐热相关PGM同工酶和与其自然电泳迁移率相近的另一同工酶的米氏常数(Km),比较了它们对37℃热处理的稳定性。结果表明,在米氏常数和热稳定性方面它们没有显著差别,因而推测PGM酶本身可能不是影响大白菜耐热性的主要因素。

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