The effect of nitrogen starvation on the levels of nucleotide triphosphates in Rhodotorula gracilis

Previous work has shown that in Rhodotorula gracilis nitrogen starvation induced by transfer of the logarithmically growing cells into a nitrogen-free medium induces a block of net RNA synthesis followed by the inhibition of prot~in synthesis. The present report deals with the changes of the purine and pyrimidine triphosphate pools in the period immediately following the transfer of the cells into the nitrogen free medium. The cells were grown aerobically for 13-15 hours at 30"C in a mineral-glucose medium containing 2 mM ammonium sulphate and 4 fLc/ml 32p orthophosphate (final orthophosphate concentration ;5~. 1O- 3M). Cells were filtered and resuspended in fresh medium, with or without ammonium sUlPhate, supplemented with 32p orthophosphate at the same specific activity. The levels of GTP, ATP, CTP, UTP were determined by extracting the cells in 1 N acetic acid, an aliquot of the lyophilized extract was spotted together with about 15 mf-Lmoles of each marker nucleotide on PEI-cellulose thin-layer plates, and nucleotides were resolved by two-dimensional chromatography according to Neuhard et al. (J. NEUHARD, E. RANDERATH e K. RANDERATH: Analytical Biochem., 13, 211, 1965). The results show that immediately before the transfer of the cells into the nitrogen free medium the ATP pool (0.6 mfLmoles/ml of culture; 1 ml of culture corresponds to about 6· 107 cells) is approximately three times larger than the pools of GTP (0.25 mf-Lmoles) and CTP (0.18 mf-Lmoles) and two times larger than the pool of UTP (0.32 mflmoles). In the period after the transfer of the cells in the new media (with and without the nitrogen source) the levels of all of the four nucleotide triphosphates pools fall by about 50-70% in the N-starved cells and by about 20-30% in the nitrogen containing culture during the first ten minutes; then they rised again, reaching after 60 minutes levels almost equal for the two different nutritional conditions. These results seem to rule out the possibility that the block of RNA accumulation in the N-starved cells is due to a lack of immediate precursors; on the other side the similarity in the levels of ATP and of the other nucleotide triphosphates indicates that under the N-starvation condition growth is not limited by the availability of high energy phosphate bonds.