摘要： 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、Sephadex G75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。

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

Abstract: A variety of factors were comparatively studied with regard to their effects on the stability of phosphoglucomutase (PGM)in Chinese cabbage.It was clearly shown that PO43-was most effective in maintaining the enzyme activity, while Tris-Cl was most ineffective.Inclusion of such possible enzyme protectants as DTT, PMSF, glucose and Mg in phosphate extraction buffer did not improve PGM stability any furthur.It was also shown that PGM enzyme was much affected by pH change, with low pH harmful and pH8.0 optimal.For these reasons, pH8.0 phosphate buffer was used throughout the process of PGM purification.The three PGM isozymes were successfully separated from the etiolated seedlings of Chinese cabbage “Baiyang”, by a series of methods from extraction, stepwise ammonium sulphate precipitation, Sephadex G75 chromatography and finally DEAE-cellulose HPLC.The Michaelis constants (Km) of these two isozymes (one is the heat-tolerance related PGM)were then determined, and their heat stability was evaluated directly by incubation at 37℃.No essential differences were found between them, indicating that the observed heat-tolerance of Chinese cabbage cannot be attributed to any individual PGM isozyme.

Key words: Phosphoglucomutase, Isozyme, Michaelis constants, Heat-tolerance, Chromatography, Chinese cabbage