Rode W, Scanlon K J, Moroson B A, Bertino J R
J Biol Chem. 1980 Feb 25;255(4):1305-11.
L1210 mouse leukemia cells were synchronized by isoleucine deprivation followed by hydroxyurea treatment. Changes of thymidylate synthetase activity both in cell extracts and in the intact cells (in situ) were followed throughout the cell cycle. The enzyme activity assayed in situ, by measuring tritium released from [5-3H]deoxyuridine, changed markedly in relation to the DNA synthesis pattern, whereas that assayed in cell extracts remained unchanged. In the S phase of the cell cycle, activity of the enzyme was much higher as measured in situ than as measured in cell extracts. Hydroxyurea (1 mM) inhibited DNA synthesis in exponentially growing L1210 cells and was found to inhibit thymidylate synthetase activity in situ. However, the enzyme activity in cell extracts was not inhibited by the drug. In cells with DNA synthesis inhibited by hydroxyurea, the pattern of change of in situ activity with time suggested product inhibition. Results of isotope-dilution analysis of the release of tritium from [5-3H]deoxyuridine in the intact cells indicated that thymidine added to the medium caused strong inhibition of thymidylate synthetase, the most likely limiting step in this pathway. These data allow the suggestion that, during cell growth, thymidylate synthetase activity is regulated by thymine nucleotide(s), and is dependent on the rate of DNA synthesis as well as the availability of the salvage pathway substrate, thymidine.
L1210小鼠白血病细胞通过异亮氨酸剥夺后用羟基脲处理进行同步化。在整个细胞周期中跟踪细胞提取物和完整细胞（原位）中胸苷酸合成酶活性的变化。通过测量从[5-³H]脱氧尿苷释放的氚来原位测定的酶活性，相对于DNA合成模式有明显变化，而在细胞提取物中测定的酶活性保持不变。在细胞周期的S期，原位测量的酶活性比在细胞提取物中测量的高得多。羟基脲（1 mM）抑制指数生长的L1210细胞中的DNA合成，并被发现抑制原位胸苷酸合成酶活性。然而，该药物不抑制细胞提取物中的酶活性。在DNA合成被羟基脲抑制的细胞中，原位活性随时间的变化模式表明存在产物抑制。对完整细胞中[5-³H]脱氧尿苷释放的氚进行同位素稀释分析的结果表明，添加到培养基中的胸苷强烈抑制胸苷酸合成酶，这是该途径中最可能的限速步骤。这些数据表明，在细胞生长过程中，胸苷酸合成酶活性受胸腺嘧啶核苷酸调节，并且依赖于DNA合成速率以及补救途径底物胸苷的可用性。
