Tan L U, Drury E J, MacKenzie R E
J Biol Chem. 1977 Feb 10;252(3):1117-22.
Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase, and formyltetrahydrofolate synthetase from porcine liver have been co-purified more than 500-fold to apparent homogeneity. The inability of three sequential chromatographic procedures followed by affinity chromatography using NADP+- or ATP-substituted Sepharose to resolve the three activities demonstrates that they are physically associated. Molecular weight estimates of the native protein by gel filtration (Mr = 150,000) and by dodecyl sulfate gel electrophoresis (Mr = 100,000) indicate that the native structure is probably a single subunit. Since only one protein band is seen on dodecyl sulfate gels, it is concluded that the three activities are properties of a single polypeptide chain. The kinetic properties of the three activities are described, the most unusual feature being the susceptibility of the cyclohydrolase to competitive inhibition by NADP+, NAD+, ATP, and folate.
猪肝中的亚甲基四氢叶酸脱氢酶、次甲基四氢叶酸环化水解酶和甲酰四氢叶酸合成酶已通过共纯化提高了500多倍，达到了明显的均一性。采用NADP⁺或ATP取代的琼脂糖进行亲和层析，随后进行三步连续层析，却无法分离这三种活性，这表明它们在物理上是相关联的。通过凝胶过滤法（Mr = 150,000）和十二烷基硫酸钠凝胶电泳法（Mr = 100,000）对天然蛋白质分子量的估计表明，其天然结构可能是一个单亚基。由于在十二烷基硫酸钠凝胶上只看到一条蛋白带，因此得出结论，这三种活性是单一多肽链的特性。本文描述了这三种活性的动力学特性，其中最不寻常的特征是环化水解酶对NADP⁺、NAD⁺、ATP和叶酸的竞争性抑制敏感。
