A single subcutaneous administration of sodium [14C] bicarbonate was given to anemic mice previously treated with acetylphenylhydrazine. 14C rapidly appeared in the arterial blood, reached a peak value in less than 3 min, and disappeared with a half‐life of about 7 min. The tracer thus administered offered a quasi pulse‐labeling technique. The radioactive intermediates and end products in the hyperplastic and hematopoietic spleen were separated by repeated chromatography on anion‐exchange resin. A relatively large amount of radioactivity appeared in carbamoylaspartate (peak value at 5 min), while definite but low radioactivity was found in dihydroorotate and orotate plus its derivatives (∑ orotate). The 14C in the uracil 5′‐nucleotide pool (∑UMP) reached a maximum at 30 min and then declined with a half‐life of about 60 min. l‐[ureido‐14C] Citrulline or l‐[guanidino‐14C] arginine induced no significant labeling of uracil nucleotides.
Steady‐state levels of carbamoyl phosphate, carbamoylaspartate, ∑ orotate, and ∑UMP were 0.12, 69.0, 5.0, and 513 nmol/g spleen. The above observations were consistent with the notion that production of carbamoyl phosphate limits the pathway, under the conditions used. When mice Were pretreated with 6‐azauridine, a potent inhibitor of orotate conversion to UMP, there was a marked enhancement in the tissue contents of the intermediates, as well as a considerable accumulation of the 14C label, thus demonstrating that intermediates do not readily permeate through cell membranes.
Kinetic analysis of the 14C label in carbamoylaspartate, together with reasonable assumptions, gave a value of 0.43 μmol × h−1× g−1 for the flow rate of the pathway. This value was close to the apparent turnover rate of ∑UMP (0.36 μmol × h−1× g−1), calculated from the half‐life of the decline of the radioactivity and the pool size of ∑UMP (0.52 μmol/g).
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