BIOSYNTHESIS OF PURINES AND PYRIMIDINES 303 



labeled ammonium chloride together with an excess of unlabeled orotic 

 acid, under conditions which permit a synthesis of polynucleotides. It 

 could be shown that the incorporation of isotope from the ammonium chlo- 

 ride was much diminished by the presence of the nonlabeled orotic acid. 

 Furthermore, the orotic acid contained a considerable amount of isotope 

 at the end of the experiment (Table X). Two possible explanations for the 

 experimental results were considered. They are represented by the equations 

 below: 



(1) NH3 — > orotic acid —y polynucleotide pyrimidines 



(2) NH3 -^ intermediate —^ polynucleotide pyrimidines 



orotic acid 



Further experiments are necessary to distinguish with certainty between 

 these two possibiUties, although there is little direct experimental evidence 

 to speak in favor of the more complicated explanation represented by equa- 

 tion (2). 



The high N^^ content of the orotic acid at the end of the experiment indi- 

 cated a considerable de novo formation from ammonia. Much more orotic 

 acid was formed than corresponded to the simultaneous de novo synthesis 

 of polynucleotide pyrimidines. It was thought, therefore, that this system 

 should prove valuable for further studies of pyrimidine biosynthesis, and 

 such studies were undertaken by Reichard and Lagerkvist.^*^* The general 

 approach was to incubate the labeled precursor and a bank of nonlabeled 

 orotic acid with liver slices, isolate the orotic acid at the end of the incuba- 

 tion, and determine the amount and distribution of isotope within the 

 molecule. In some cases dilution experiments were also carried out, in 

 which the unlabeled precursor was incubated in the system together with 

 N^Mabeled ammonium chloride and orotic acid. Again the amount and dis- 

 tribution of N'* in the orotic acid was determined at the end of the experi- 

 ment, and in one case the precursor was also isolated and analyzed for N'^ 



The degradation of orotic acid is outlined in Fig. 11. After hydrogenation 

 to dihydroorotic acid, the ring was spht by acid treatment. Nitrogen 1 was 

 obtained as ammonia nitrogen 3 plus carbons 4 to 7 as aspartic acid. The 

 sum of carbons 6 and 7 was determined as CO2 by ninhydrin decarboxyla- 

 tion of the aspartic acid, and carbons 4 plus 5 were obtained by difference. 

 Carbon 2 was determined as urea by a separate degradation of the orotic 

 acid with permanganate. The reliability of the method was tested by deg- 

 radation of orotic acid labeled with N^^ in position 3. 



The precursors investigated were N'^-labeled ammonium chloride, bi- 

 carbonate-C^^ L-aspartate-N'*, L-aspartate-l,4-C'^ L-aspartate-2,3-C"and 

 L-ureidosuccinie acid (labeled with N^^ in the nitrogen atom which, after 

 ring-closure, corresponds to position 3 of orotic acid). Dilution experiments 



