Höglund L, Pontis E, Reichard P
Department of Biochemistry I, Medical Nobel Institute, Karolinska Instiutet, Stockholm, Sweden.
Eur J Biochem. 1991 Feb 26;196(1):239-45. doi: 10.1111/j.1432-1033.1991.tb15810.x.
V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.
通过在逐步增加药物浓度的条件下连续培养，使V79仓鼠细胞对羟基脲产生抗性。克隆了两个细胞系，分别对0.4 mM（V79/H0.4）和4 mM（V79/H4）羟基脲具有抗性，其可溶性核糖核苷酸还原酶活性分别增加了5倍和20倍。我们研究了酶量的增加如何影响核糖核苷酸还原酶的原位活性和脱氧核糖核苷酸代谢，特别是嘧啶脱氧核糖核苷及其5'-磷酸之间的底物循环。还原酶的原位活性仅适度升高（V79/H4细胞中为1.3倍）。在完全抗性的细胞系中，dATP的稳态水平增加了4倍，dTTP增加了2倍。这些核苷酸是还原酶的负别构效应物，我们认为增加的库抑制了该酶，从而使还原酶的原位活性仅略微升高。多余的脱氧核糖核苷酸通过底物循环以胸苷和脱氧胞苷的形式从细胞中排出。这些数据支持并扩展了我们先前提出的通过核糖核苷酸还原酶的别构性质和连接脱氧核糖核苷酸补救与降解的底物循环来调节脱氧核糖核苷酸合成的模型。
