Bachmann B, Follmann H
Arch Biochem Biophys. 1987 Jul;256(1):244-52. doi: 10.1016/0003-9861(87)90442-5.
Thymidylate synthase and dihydrofolate reductase are peak enzymes that accompany the S phase of the unicellular green algae, Scenedesmus obliquus, and are both overproduced in the presence of 5-fluorodeoxyuridine. Such overproducing cultures have served for enzyme isolation and characterization. It has not been possible to separate the two enzyme activities by several methods of protein fractionation, including affinity chromatography on specific immobilized ligands (fluorodeoxyuridylate or N10-formylfolate); both were enriched in parallel approximately 400-fold from algal extracts. The most highly purified samples are of low stability in solution. Enzyme activities are inhibited by methotrexate, 5-fluorodeoxyuridylate, and arabinouridylate but not by hydroxyurea; FdUMP inhibition is fully reversed after removal of the nucleotide. Sedimentation in sucrose gradients (Mr 100,000) and electrophoresis in denaturing polyacrylamide gels (Mr 50,000) suggest that the protein structure resembles more the dimeric, bifunctional thymidylate synthase-dihydrofolate reductase of protozoan species than the separate enzymes found in bacteria and animal cells.
胸苷酸合成酶和二氢叶酸还原酶是单细胞绿藻斜生栅藻S期的峰值酶，在5-氟脱氧尿苷存在时均会过量产生。这种过量产生的培养物已用于酶的分离和特性鉴定。通过几种蛋白质分级分离方法，包括在特定固定化配体（氟脱氧尿苷酸或N10-甲酰叶酸）上进行亲和色谱，都无法分离这两种酶活性；两者在藻提取物中平行富集约400倍。纯化程度最高的样品在溶液中的稳定性较低。酶活性受到甲氨蝶呤、5-氟脱氧尿苷酸和阿拉伯糖基尿苷酸的抑制，但不受羟基脲的抑制；去除核苷酸后，FdUMP抑制作用完全逆转。蔗糖梯度沉降（分子量100,000）和变性聚丙烯酰胺凝胶电泳（分子量50,000）表明，该蛋白质结构更类似于原生动物物种的二聚体双功能胸苷酸合成酶-二氢叶酸还原酶，而不是细菌和动物细胞中发现的单独的酶。
