Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay.
Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay.
复制标题
鉴定寡胸苷酸作为大肠杆菌 DNA 光裂合酶的新简单底物及其在快速分光光度酶测定中的应用。
DOI：
10.1021/bi00329a008
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Sancar,A
中科院分区：
文献类型：
--
作者：
Jorns,MS;Sancar,GB;Sancar,A
方法.克隆E.通过别处描述的方法分离大肠杆菌DNA光裂合酶（Sanear等人，1984年a）。将酶的浓缩储备溶液在-80或-20 ℃下储存在含有50 mMNaCl、1.0mMEDTA、10 mMDTT和50%甘油的50 mMTris（pH7.4）中.在带塞石英半微量比色皿（250 μ L/比色皿）中照射oligo（dT），8（5.9 × 10 - 5 M）在10 mM Tris-HCl，pH7.2中的储备溶液，该溶液含有0.5mM EDTA。将比色皿水平放置在光学面上，并在4 ℃下从上方照射6分钟，同时摇动（Tektator V摇动器），距离两个杀菌灯（西尔瓦尼亚G15 T8，15 W）5 cm。通过寡聚物（dT）n的浓缩溶液在290或295 nm处或稀释溶液在260 nm处的吸光度降低来监测二聚体形成。同样的程序用于与其他寡胸苷酸的照射
Methods. Cloned E. coli DNA photolyase was isolated by a procedure described elsewhere (Sanear et al., 1984a). Concentrated stock solutions of enzyme were stored at-80 or-20 C in 50 mM Tris, pH 7.4, containing 50 mM NaCl, 1.0 mM EDTA, 10 mM DTT, and 50% glycerol. Except where noted, all handling of the enzyme was done under yellow light.Stock solutions of oligo (dT), 8 (5.9 X 10" 5 M) in 10 mM Tris-HCl, pH 7.2, containing 0.5 mM EDTA were irradiated in stoppered quartz semimicro cuvettes (250 juL/cuvette). The cuvettes were laid horizontally on an optical face and irradiated from above for 6 min at 4 C with shaking (Tektator V shaker) at a distance of 5 cm from two germicidal lights (Sylvania G15T8, 15 W). Dimer formation was monitored by decreases in absorbance at 290 or 295 nm for concentrated solutions of oligo (dT)„or at 260 nm for dilute solutions. The same procedure was used for irradiation with other oligothymidylates