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 (1mM) 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.
|Number of pages
|Journal of Biological Chemistry
|Published - 1980
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology