Chen G X, Zolg J W
Department of Molecular Biology, Biomedical Research Institute, Rockville, Maryland 20852.
Mol Pharmacol. 1987 Dec;32(6):723-30.
The bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum has been purified to homogeneity using a sequence of separation steps including phenyl-Superose, gel filtration, dye affinity matrix, hydroxyapatite, and anion exchange chromatography. The specific activity of dihydrofolate reductase increased approximately 24,000-fold from 3.3 units mg-1 protein to 79,000 units mg-1 protein after five successive chromatographic steps with a yield of 31%. Both enzyme activities coeluted as a symmetric peak in highly purified preparations, indicating the existence of a bifunctional enzyme complex in P. falciparum. The apparent molecular weight of the native complex was approximately 120,000 as determined by gel filtration. When individual fractions of the anion exchange column were subject to polyacrylamide electrophoresis under denaturing conditions, the increase in intensity of a single band correlated with the amount of both the thymidylate synthase and dihydrofolate reductase activity. Further purification led to an electrophoretically pure protein (yield 2.6%) with an apparent molecular weight of 67,000, suggesting that the bifunctional enzyme complex from P. falciparum is composed of two subunits of identical size and charge.
利用一系列分离步骤，包括苯基超琼脂糖、凝胶过滤、染料亲和基质、羟基磷灰石和阴离子交换色谱，已将来自人类疟原虫恶性疟原虫的双功能胸苷酸合酶 - 二氢叶酸还原酶复合物纯化至同质。经过五个连续的色谱步骤后，二氢叶酸还原酶的比活性从3.3单位毫克-1蛋白质增加到79,000单位毫克-1蛋白质，增加了约24,000倍，产率为31%。在高度纯化的制剂中，两种酶活性以对称峰的形式共洗脱，表明恶性疟原虫中存在双功能酶复合物。通过凝胶过滤测定，天然复合物的表观分子量约为120,000。当阴离子交换柱的各个馏分在变性条件下进行聚丙烯酰胺电泳时，单一条带强度的增加与胸苷酸合酶和二氢叶酸还原酶活性的量相关。进一步纯化得到了一种表观分子量为67,000的电泳纯蛋白（产率2.6%），表明来自恶性疟原虫的双功能酶复合物由两个大小和电荷相同的亚基组成。
