Regulation of thymidylate synthetase in mouse leukemia cells (L1210).
Regulation of thymidylate synthetase in mouse leukemia cells (L1210).
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小鼠白血病细胞 (L1210) 中胸苷酸合成酶的调节。
DOI：
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发表时间：
1980
影响因子：
4.8
通讯作者：
J. Bertino
中科院分区：
文献类型：
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作者：
W. Rode;K. Scanlon;B. Moroson;J. Bertino
L1210小鼠白血病细胞同步化的异亮氨酸剥夺，然后用羟基脲处理。胸苷酸合成酶活性的变化，在细胞提取物和完整的细胞（原位）在整个细胞周期。通过测量从[5- 3 H]脱氧尿苷中释放的氚，原位测定的酶活性与DNA合成模式有关，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，酶的活性比在细胞提取物中测得的高得多，在原位测量。在指数生长的L1210细胞中，羟基脲（1 mM）抑制DNA合成，并发现原位抑制胸苷酸合成酶活性。然而，细胞提取物中的酶活性不被药物抑制。在DNA合成受到羟基脲抑制的细胞中，原位活性随时间变化的模式表明产物抑制。完整细胞中[5- 3 H]脱氧尿苷释放氚的同位素稀释分析结果表明，添加到培养基中的胸苷对胸苷酸合成酶产生强烈抑制，这是该途径中最可能的限制步骤。这些数据表明，在细胞生长期间，胸苷酸合成酶活性受胸腺嘧啶核苷酸调节，并取决于DNA合成速率以及补救途径底物胸苷的可用性。
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.