Zhang Q M, Fujimoto J, Yonei S
Laboratory of Radiation Biology, Faculty of Science, Kyoto University, Japan.
Int J Radiat Biol. 1995 Dec;68(6):603-7. doi: 10.1080/09553009514551601.
To identify a repair enzyme for 5-formyluracil (5-FU) caused by ionizing radiation in DNA, we used a radiolabelled product-release assay for this thymine-damaged substrate. Double-stranded poly(dA-dT)-poly(dA-dT) was radiolabelled by nick translation with [2-14C]-thymidine triphosphate. The DNA was irradiated with X-rays and incubated with cell extract from mouse liver. Radiolabelled products released from the irradiated DNA into an ethanol-soluble fraction were analysed by reversed-phase hplc. Released 5-FU was detected as a free base during reaction with the cell extract. 5-Formyl-2'-deoxyuridine was not detected in the ethanol supernatant. Boiling the extract at 97 degrees C for 15 min completely abolished its ability to release 5-FU. Similar enzymatic activity was observed with rat liver extract. These results demonstrated that mammalian cells have enzymatic activity to release 5-FU from DNA.
为了鉴定DNA中由电离辐射导致的5-甲酰基尿嘧啶（5-FU）的修复酶，我们针对这种胸腺嘧啶损伤的底物使用了放射性标记的产物释放测定法。双链聚（dA-dT）-聚（dA-dT）通过用[2-¹⁴C]-胸苷三磷酸进行缺口平移进行放射性标记。DNA用X射线照射，并与小鼠肝脏的细胞提取物一起孵育。通过反相高效液相色谱分析从受辐照DNA释放到乙醇可溶级分中的放射性标记产物。在与细胞提取物反应期间，释放的5-FU被检测为游离碱。在乙醇上清液中未检测到5-甲酰基-2'-脱氧尿苷。将提取物在97℃下煮沸15分钟完全消除了其释放5-FU的能力。用大鼠肝脏提取物观察到类似的酶活性。这些结果表明哺乳动物细胞具有从DNA释放5-FU的酶活性。
