Abstract
A simple micromethod was established for the accurate measurement of dTMP synthase activity in rat liver crude extracts. The reaction product of dTMP synthase assay, i.e., tritiated water, released from (5-3H)deoxyuridine 5′-monophosphate(dUMP), was separated in one-step with, 100% KOH absorption from (5-3H)deoxyuridine (dUrd) which is the side-product of dephosphorylation of (5-3H)dUMP during the enzyme reaction. Tritiated water was trapped in three droplets of 100% KOH deposited on the underside of the vessels' lids, while (3H)dUrd remained in the bottom of vessels after absorption of (5-3H)dUMP from the reaction mixture by charcoal treatment. Under standard assay conditions in the crude extracts of rat liver, the specific activity of dTMP synthase and dUMP phosphatase were 0.092±0.002 and 0.351±0.013 nmol/hr/mg protein,respectively. This method was also adapted for dTMP synthase assay in human lung cancer cells. The major advantages of this method are the elimination of the phosphatase activity which interferes with the estimation of dTMP synthase activity,one-step separation of 3H2O, high sensitivity, high reproducibility and low requirement of tissue.
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Hashimoto, Y., Shiotani, T., Fujita, J. et al. 141 Improved assay method of dTMP synthase in rat liver and its application to human lung cancer cells. Pediatr Res 24, 134 (1988). https://doi.org/10.1203/00006450-198807000-00165
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DOI: https://doi.org/10.1203/00006450-198807000-00165