Enhancement of Intracellular 5‐Phosphoribosyl 1‐Pyrophosphate Levels as a Major Factor in the 6‐Azauridine‐Induced Stimulation of Carbamoyl Phosphate Synthesis in Mouse Spleen Slices

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Abstract
Brief exposure to 6‐azauridine stimulates the production of carbamoyl phosphate for de novo pyrimidine biosynthesis in vitro in slices of haematopoietic spleen from anaemic mice (preceding paper). In studies of the underlying mechanism for this response we turned our attention to changes in the level of substrates and effectors for carbamoyl‐phosphate synthetase II. Intermediates of the orotic acid pathway and 6‐azauridine had little effect on the synthetase activity in vitro. 6‐Azauridine 5′‐monophosphate (6‐AzaUMP) stimulated synthetase II, possibly in an allosteric manner. However, in view of the potency as an activator and the tissue levels, 6‐azaUMP may be only partially responsible for the stimulation. Adenine nucleotide levels in the tissue showed only minor changes after brief exposure (15 min) to 6‐azauridine. The level of UTP and UDP, potent inhibitors for synthetase II, showed no significant change. The level of 5‐phosphoribosyl 1‐pyrophosphate (PPRibP), a potent positive effector for the synthetase II, showed a more than 1.5‐fold increase after 15 min. The relative importance of these factors was evaluated by assay of the synthetase, partially purified from mouse spleen, under simulated conditions in vitro. The results indicated that the enhanced level of PPRibP played a major role in increasing the production of carbamoyl phosphate. In Ehrlich ascites cells in vitro, where 6‐azauridine did not increase carbamoyl phosphate production, the basal PPRibP level was high (range over 0.1 mM) and the changes in the level, brought about by the analogue, were relatively small.