PURIFICATION AND PROPERTIES OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE
PURIFICATION AND PROPERTIES OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE
复制标题
DOI：
10.1021/bi00695a006
复制
发表时间：
1975-01-01
期刊：
影响因子：
2.9
通讯作者：
BURCHALL, J
中科院分区：
文献类型：
--
作者：
BACCANARI, D;PHILLIPS, A;BURCHALL, J
用包括甲氨蝶呤亲和柱层析的方法，从耐甲氧氨嘧啶的大肠杆菌(RT 500)菌株中纯化了40倍的二氢叶酸还原酶，使其表观均一。根据氨基酸组成、沉降速度和十二烷基硫酸钠凝胶电泳法测定该酶的相对分子质量分别为17680、17470和18300。通过凝胶过滤可以将具有低比活性的聚集态的酶从单体中分离出来；用巯基乙醇或二硫苏糖醇处理聚合体可以提高酶的活性并使单体再生。二氢叶酸还原酶[5，6，7，8-四氢叶酸：NADP+氧化还原酶(EC 1.5.1.3)]是几种重要药物的靶酶。我们以前已经证明，细菌来源的二氢叶酸还原酶和哺乳动物来源的二氢叶酸还原酶可以根据它们与一系列二氢叶酸小分子异构体的相对结合能力来区分。这些结合上的差异解释了这些药物如何作为有效和无毒的抗菌剂发挥作用，尽管它们的靶标对宿主和寄生虫都是共同的(Burchall和Hitchings，1965)。
Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resis-tant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatogra-phy. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470, and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis.Dihydrofolate reductase[5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase (EC 1.5. 1.3)] is the target enzyme of several important drugs. We have previously shown that dihydrofolate reductases from bacterial and mammalian sources can be distinguished from each other on the basis of their relative ability to bind a series of smallmolecule an-alogues of dihydrofolate. These differences in binding ex-plain how the drugs can function as potent and nontoxic an-tibacterials even though their target is common to both host and parasite (Burchall and Hitchings, 1965).