Haber D A, Beverley S M, Kiely M L, Schimke R T
J Biol Chem. 1981 Sep 25;256(18):9501-10.
We have studied a line of 3T6 mouse embryo fibroblasts grown in progressively increasing concentrations of methotrexate. Resistance of cells to low concentrations of the inhibitor (less than 5 X 10(-5) M) is attributed to selective multiplication of the genes coding for dihydrofolate reductase and the resulting elevation of enzyme content. Cells isolated at a higher methotrexate concentration (4 X 10(-4) M) contain high levels of a dihydrofolate reductase with a reduced affinity for methotrexate. The altered dihydrofolate reductase exhibits a 270-fold reduction in binding affinity for methotrexate as measured by equilibrium dialysis (Kd = 5.4 X 10(-8) M versus 2 X 10(-10) M for the wild type enzyme). While binding to NADPH is unchanged, the Km for dihydrofolate is increased 3-fold over wild type enzyme and the turnover number for the reduction of dihydrofolate to tetrahydrofolate is decreased 20-fold. The altered dihydrofolate reductase shows a broader and more predominant acidic peak in its pH profile for this reaction. The molecular weights of the altered and wild type enzymes are identical as determined by sodium dodecyl sulfate-gel electrophoresis, but 2-dimensional electrophoresis reveals a significant basic shift in the migration of the altered enzyme. Studies with various folic acid analogs suggest that modifications involving the para-aminobenzoyl moiety of the inhibitor molecules are associated with the most dramatic differential binding between the altered and wild type dihydrofolate reductases.
我们研究了在甲氨蝶呤浓度逐渐增加的条件下培养的3T6小鼠胚胎成纤维细胞系。细胞对低浓度抑制剂（小于5×10⁻⁵M）的抗性归因于编码二氢叶酸还原酶的基因的选择性增殖以及由此导致的酶含量升高。在较高甲氨蝶呤浓度（4×10⁻⁴M）下分离的细胞含有高水平的二氢叶酸还原酶，其对甲氨蝶呤的亲和力降低。通过平衡透析测量，改变后的二氢叶酸还原酶对甲氨蝶呤的结合亲和力降低了270倍（野生型酶的Kd = 2×10⁻¹⁰M，而改变后的酶为5.4×10⁻⁸M）。虽然与NADPH的结合未改变，但二氢叶酸的Km值比野生型酶增加了3倍，将二氢叶酸还原为四氢叶酸的周转数降低了20倍。在该反应的pH谱中，改变后的二氢叶酸还原酶显示出更宽且更突出的酸性峰。通过十二烷基硫酸钠 - 凝胶电泳测定，改变后的酶和野生型酶的分子量相同，但二维电泳显示改变后的酶迁移有明显的碱性位移。对各种叶酸类似物的研究表明，涉及抑制剂分子对氨基苯甲酰部分的修饰与改变后的和野生型二氢叶酸还原酶之间最显著的差异结合有关。
