Comparison of incorporation and metabolism of RNA pyrimidine nucleotide precursors in leaf tissues.

Abstract

The pathway of synthesis of cytidylic and uridylic acids has been tentatively established in certain microbial and animal cells according to recent reviews (14, 35, 28). The sequence of reactions is probably as follows: CO, + ATP + NH3 > carbamyl -H.,O aspartate phosphate --> carbamyl aspartate > di-2 H PRPP3 hydroorotic acid orotic acid > orotidine-5'-phosphate -> UMP + CO2. In yeast, UMP was further converted to UTP by kinases requiring ATP (23). Amination of uridine nucleotides to form cytidine nucleotides involved participation of glutamine in mammalian cells (18, 34) and NH3 in Escherichia coli (21). Neither uracil or uridine, cytosine or cytidine, are normal intermediates in synthesis of uridylic and cytidylic acids. The only pyrimidine involved is orotic acid, according to this pathway. In higher plants, uracil, uridine, and cytidine are incorporated into RNA (2, 8, 15), although incorporation of cytosine has apparently not been reported. Anabolism of -uracil may occur either by addition of ribose from ribose-1-phosphate to produce uridine (11) or by conversion directly to UMP upon reaction with PRPP (11, 13). Uridine was converted to UMP in the presence of ATP and a uridine kinase (11). The ,enzymes responsible for cytidine utilization have ap_parently not been extensively studied. Considerably less is known of the pathway of pyrimidine nucleotide synthesis in higher plants. In a preliminary communication Kapoor and Waygood (19) reported in wheat embryos the presence of the enzyme converting orotic acid to OMP, orotidine-5'phosphate pyrophosphorylase, and of dihydroortic de-hydrogenase, which forms orotate from dihydroorotate. In a similar brief report, King and Wang (20) showed that C14 from both uracil and aspartic acid labelled pyrimidines of wheat leaves. Neumann and Jones (25) rccently studied aspartic acid transcarbamylase, and demonstrated several of its properties. Buchowicz and Reifer (7) found orotic acid to be converted to UMP, CMP, uridine, and uracil in wheat seedling leaves. Carbamyl aspartate also labelled these compounds and, in addition, orotic acid (30). This work suggests a pathway of nucleotide synthesis similar to that in other organisms. However, the latter workers reported that the conversion of orotic acid to UMP involved uridine as an intermediate product (9), and were unable to detect any OMP in their wheat leaves. Buchowicz (8) also reported that uracil could be incorporated directly into the nucleotides of RNA in wheat seedling homogenates without first labelling UMP and CMP. A direct binding of uracil by a polynucleotide acceptor was suggested to explain this surprising result. The present experiments were undertaken to provide more evidence as to some of the reactions involved in synthesis of uridylic and cytidylic acids in higher plants and to extend the results to plants other than wheat. Quantitative comparisons of the rate of incorporation of certain precursors into RNA were made and several metabolites were identified, including OMP.