Chimploy K, Tassotto M L, Mathews C K
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, USA.
J Biol Chem. 2000 Dec 15;275(50):39267-71. doi: 10.1074/jbc.M006233200.
While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O(2) is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O(2) is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.
在研究哺乳动物细胞中脱氧核糖核苷三磷酸（dNTP）库显著自然不对称性的基础时，我们观察到在2%氧气氛围中培养V79仓鼠肺细胞会导致dATP、dGTP和dTTP库扩大2 - 3倍，而dCTP则相应减少。其他人也有类似观察结果，并提出由于核糖核苷酸还原酶中催化必需的氧桥连铁中心的形成需要O₂，低氧张力下dCTP的消耗是对核糖核苷酸还原酶直接或间接作用的结果。我们使用重组小鼠核糖核苷酸还原酶和一种能够同时监测所有四种核苷酸底物还原情况的测定方法，对氧限制影响核糖核苷酸特异性这一假设进行了测试。在厌氧箱中对该酶进行预孵育和测定仅导致部分活性丧失。因此，我们先用羟基脲处理该酶，然后去除羟基脲并使其暴露于不同氧含量的环境中。羟基脲处理使活性完全丧失，而暴露于空气中后活性几乎完全恢复。根据在不同氧张力下恢复的活性标准，我们发现CDP的还原对氧消耗并不特别敏感；然而，GDP的还原则特别敏感。O₂对再激活产生差异反应的基础尚不清楚，但显然它不涉及该酶不同别构形式再激活速率的变化，也不涉及在降低的氧张力下对别构效应物反应的改变。
