PYRIMIDINE NUCLEOTIDE-METABOLISM IN RAT HEPATOCYTES - EVIDENCE FOR COMPARTMENTATION OF NUCLEOTIDE POOLS

Pyrimidine nucleotide metabolism in rat hepatocytes was studied by mea surement of the labelling kinetics of the various intermediates after double labelling with [C-14]orotic acid and [H-3]cytidine, the precurs ors for the de novo and the salvage pathways respectively. For the uri dine nucleotides, differences were found for the C-14/H-3 ratios in th e UDP-sugars, in UMP (of RNA) and in their precursor UTP, suggesting t he existence of separated flows of the radioactive precursors through the de novo and the salvage pathways. Higher ratios in the UDP-sugars, which are synthesized in the cytoplasm, and a lower ratio in UMP (of RNA) relative to the C-14/H-3 ratio in UTP indicated that UTP derived from orotic acid is preferentially used for the cytoplasmic biosynthes is of the UDP-sugars. Uridine, derived from cytidine, is preferentiall y used for the nuclear-localized synthesis of RNA. In contrast to thes e findings, the C-14/H-3 ratios in the cytidine derivatives CMP-NeuAc and CMP (of RNA), and in the liponucleotides CDP-choline and CDP-ethan olamine, were all lower than that in the precursor CTP. This. indicate s a preferential utilization of the salvage-derived CTP for the synthe sis of the liponucleotides as well as for RNA and CMP-NeuAc. Similar c onclusions could be drawn from experiments in which the intracellular amounts of several uridine- and cytidine-nucleotide-containing derivat ives were increased by preincubating the hepatocytes with unlabelled p yrimidine nucleotides or ethanolamine. Based on these data, we propose a refined model for the intracellular compartmentation of pyrimidine nucleotide biosynthesis in which three pools of UTP are distinguished: a pool of de novo-derived molecules and a pool of salvage-derived mol ecules, both of which are channelled to the site of utilization; in ad dition an 'overflow' pool exists, consisting of molecules having escap ed from channelling. An overflow pool could also be distinguished for CTP, but no discrimination between de novo and salvage-derived molecul es could be made.