Isotope-dilution analysis of rate-limiting steps and pools affecting the incorporation of thymidine and deoxycytidine into cultured thymus cells

1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0–30μm) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (`isotope-dilution plots'), values were obtained for (a) the Vmax. of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the Vmax. and pool concentration at a critical thymidine concentration (approx. 5μm). The bimodality was removed by amethopterin. The Vmax. determined with deoxy[U-14C]cytidine was approximately equal to the sum of the Vmax. determined with deoxy[5-3H]cytidine and the Vmax. determined with [Me-3H]thymidine at thymidine concentrations above 5μm. 3. The thymidine competitor pool at thymidine concentrations above 5μm was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5μm. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16μm) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5μm, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5μm, thymidine kinase limits the rate of labelling with [Me-3H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4μm). At thymidine concentrations greater than 5μm, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.