Duffy T H, Beckman S B, Sato J K, Nagae H, Vitols K S, Huennekens F M
Adv Enzyme Regul. 1985;23:3-12. doi: 10.1016/0065-2571(85)90037-8.
Dihydrofolate reductase, purified to homogeneity (as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), from a subline of L1210 murine leukemia cells resistant to 10(-6) M methotrexate, was resolved into two principal forms (1 and 2) by polyacrylamide gel electrophoresis at pH 8.3 or isoelectric focusing. In the latter procedure, these forms had pI values of 7.4 and 8.2, respectively; both stained for protein and catalytic activity. Form 1 appears to be a single component, comprising ca. 10% of the total protein and at least 20% of the total catalytic activity. It is also more sensitive to inhibition by MTX, more heat-stable, and less susceptible to activation than form 2. Multiple components of 2 were observed by narrowing the pH range in isoelectric focusing, and further resolution was achieved by urea denaturation. Substrate and inhibitor complexes of 1 and 2, differentiated by polyacrylamide gel electrophoresis or isoelectric focusing, provided information about the ability of the enzyme to undergo conformational changes. Interconversion of 1 with one of the components of 2 may also involve conformational isomerism. These conclusions are consistent with the well-known ability of eukaryotic dihydrofolate reductases to exhibit increased catalytic activity (attributed to transformations to more open conformations) when treated with salts, chaotropes, or cysteine-modifying agents. Treatment of the L1210/R6 enzyme preparation with one of these activating agents, 5,5'-dithiobis(2-nitrobenzoic acid), derivatized both 1 and 2 (changing their pI values to 7.3 and 6.9, respectively) and altered the enzyme such that stoichiometric inhibition for MTX was observed.
从对10⁻⁶ M甲氨蝶呤具有抗性的L1210小鼠白血病细胞亚系中纯化至同质（通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断）的二氢叶酸还原酶，在pH 8.3下通过聚丙烯酰胺凝胶电泳或等电聚焦被解析为两种主要形式（1和2）。在后者的方法中，这些形式的等电点值分别为7.4和8.2；两者均对蛋白质和催化活性染色。形式1似乎是单一成分，约占总蛋白的10%，至少占总催化活性的20%。它对甲氨蝶呤抑制更敏感，更耐热，并且比形式2更不易被激活。通过在等电聚焦中缩小pH范围观察到形式2的多个成分，并且通过尿素变性实现了进一步的解析。通过聚丙烯酰胺凝胶电泳或等电聚焦区分的1和2的底物和抑制剂复合物提供了有关酶进行构象变化能力的信息。1与2的一种成分之间的相互转化也可能涉及构象异构。这些结论与真核二氢叶酸还原酶在用盐、离液剂或半胱氨酸修饰剂处理时表现出增加的催化活性（归因于转变为更开放的构象）的众所周知的能力一致。用这些激活剂之一5,5'-二硫代双（2-硝基苯甲酸）处理L1210/R6酶制剂，使1和2都衍生化（它们的等电点值分别变为7.3和6.9），并改变了酶，从而观察到对甲氨蝶呤的化学计量抑制。
