1. Slices of spleen from anaemic mice were incubated with [14C]
bicarbonate in the presence and absence of
6-azauridine and the amounts of 14C that entered the de novo
pyrimidine biosynthetic pathway were assessed and compared. Compounds analyzed included carbamoylaspartate,
dihydroorotate, orotate plus its derivatives,
acid-soluble
uracil and
cytosine 5'-nucleotides,
nucleic acid pyrimidines, free
pyrimidine bases and
nucleosides. As the intracellular levels of
carbamoyl phosphate and
acid-soluble
deoxyribonucleotides are known to be relatively low, the radioactivities of these compounds were not measured. Degradation of labelled
uridine was limited in this tissues, therefore the radioactivity of degradative products of
pyrimidines was not considered. 2. When the slices were incubated with 0.5 mM
6-azauridine for 10 min and then with [14C]
bicarbonate for an additional 10 min and 30 min, the sum of radioactivity found in the above compounds, which represents the total amount of 14C that entered the
pyrimidine pathway, was 2.1 and 2.3 times greater than when the tissue slices were incubated in the absence of the analogue. 3. When the 14C distribution among the
carbon atoms of the molecules of labelled carbamoylaspartate and
uracil was investigated, we found that more than 90% of the total 14C in these compounds derived directly from
carbamoyl phosphate and the remaining portion was from
aspartate, either in the presence or absence of
6-azauridine. 4. There was no indication that
6-azauridine altered [14C]
bicarbonate permeation through the cell membrane or its intracellular metabolism. 5. These results, along with the pattern of early intermediate accumulation seen in the presence of
6-azauridine, indicate that
6-azauridine stimulates the production of
carbamoyl phosphate for the
pyrimidine biosynthetic pathway in the mouse spleen. 6. Of the radioactive early intermediates which accumulated, only orotate, its derivatives (
orotidine and
orotidine 5'-monophosphate) or both appeared in the medium, presumably the result of leakage through the cell membranes. 7. Stimulation of the
pyrimidine pathway was not observed in the case of Ehrlich
ascites tumour cells incubated under similar conditions with
6-azauridine.