Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

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Abstract
The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (C. pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides via the orotate pathway in C. pepo included: demonstration of the incorporation of [14C]NaHCO3, [14C]carbamylaspartate, and [14C]orotic acid into uridine nucleotides; the isolation of [14C]orotic acid when [14C]NaHCO3 and [14C]carbamylasparate were used as precursors; the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts. Regulation of the activity of the orotate pathway by end product inhibition was demonstrated in the intact cells of excised roots by measuring the influence of added pyrimidine nucleosides on the incorporation of [14C]NaHCO3 into uridine nucleotides. The addition of either uridine or cytidine inhibited the incorporation of [14C]NaHCO3 into uridine nucleotides by about 80%. The observed inhibition was demonstrated to be readily reversible upon transfer of the roots to a nucleoside-free medium. Experiments employing various radiolabeled precursors indicated that one or both of the first 2 enzymes in the orotate pathway are the only site(s) of regulation of physiological importance.