Thymidine uptake, its phosphorylation, and incorporation into DNA were studied in a fast-growing sugar-beet (Beta vulgaris L.) cell suspension. A high rate and specificity of thymidine uptake were observed: total uptake reached a steady-state level for 3 min. The average kinetic constants for thymidine uptake were calculated for eight-day-old cells at 20°C as K M = 25 μM and V max = 11.6 pmol/(min mg fr wt). The values of K M for thymidine phosphorylation in vivo (53.2 μM) and K M for thymidine kinase (EC 18.104.22.168), which we purified earlier from broad bean seedlings, were of the same order of magnitude. The kinetics of thymidine phosphorylation in vivo displayed two distinct phases, which were determined by external thymidine concentration. Above 100 μM thymidine, the rate of the process tended to rise, indicating the possible involvement of another mechanism for thymidine phosphorylation, most likely with the participation of nonspecific nucleoside phosphotransferase (NPT; EC 22.214.171.124). A further stage of thymidine salvage, its incorporation into DNA, occurred with a high affinity for thymidine phosphates; K M = 2.8 μM. The presence of other nucleosides (uridine or a high concentration of adenosine) in the medium markedly inhibited thymidine uptake. Nevertheless, these nucleosides did not diminish the percentage of thymidine phosphates of total thymidine uptake, which pointed to the specificity of thymidine phosphorylation and the insignificance of NPT activity. The analogue of thymidine, 5"-amino-2",5"-dideoxythymidine, known as thymidine kinase inhibitor, had no effect on thymidine uptake. The data presented provide evidence that the main route of the thymidine salvage in fast-growing sugar-beet suspension engages thymidine kinase, and NPT is activated only when nucleosides “flood” the cell.
Russian Journal of Plant Physiology – Springer Journals
Published: Oct 13, 2004
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