Isotopic discrimination between ³H and ¹⁴C in vitro during enzymatic phosphorylation of labelled thymidine

We compare the phosphorylation of 6‐³H‐TdR and 2‐¹⁴C‐TdR to TMP, TDP and TTP by tissue extracts and to TMP by purified kinase preparations. The rate of phosphorylation of 6‐³H‐TdR is only 0.6 to 0.8 that of 2‐¹⁴C‐TdR. The same isotopic discrimination exists between methyl‐labelled thymidines. In mixtures of ³H‐TdR and ¹⁴C‐TdR incubated together with a liver extract, the isotopic ratio is lower in the products than in the substrate. Degradation products of ³H‐TdR do not seem to interfere with enzymatic activity. This isotopic discrimination is tentatively ascribed to an isotopic effect of ³H which is mainly detectable at the high substrate/enzyme ratios prevailing in the in vitro incubation conditions.