304 



PETER REICHARD 



HNf 6 ,CH 

 0^C^3 IC-COOH 



H 



Orotic acid 



Pt 



O 



= C^3 J.CH-COOH 

 H 



HCl 



Dihydroorotic acid 



6C00H 

 I 



CH2 1 



1 3 + NH3 

 CH-NH2 

 I 

 7C00H 



Aspartic acid 



6C00H 

 I 



CH2 

 I 

 CH-NHj 



ninhydrin 



C + 7 

 2CO2 



7C00H 



Aspartic acid 

 Fig. 11. Degradation of orotic acid according to Reichard and Lagerkvist.'" 



TABLE XI 

 Precursors in the Biosynthesis of Orotic Acid in Liver Slices"''"^ 



Isotopic precursor* 



1 mM. L-aspartate-N'^ 



0.5 mM. L-aspartate-1 ,4-C'^ 



0.5 mM. L-aspartate-2,3-C". . . 



8 mM. NaHC'^Os 



0.2 mM. L-ureidosuccinate-N'^. 



Hours 

 incuba- 

 tion 



Isotope concentration'^ in 



Orotic 

 acid 



0.059<i 

 0.25 

 129 

 0.146 

 2.70 



Ni 



0.012 



0.10 



N3 



0.106 



5.76 



C2 



0.04 



11 



0.534 



Ce + 



0.53 

 56 



C4 + 



0.04 

 261 



" In each experiment the isotopic precursor was incubated with slices from 3 livers together with 15 mg. 

 orotic acid. 



*" Aspartate- N'5 = 32 atom % excess; aspartate-C'^ = 18 atom % excess; NaHC'^Oa = 36 atom % excess, 

 ureidosuccinate-N'* = 16 atom % excess; aspartate-C* = 8100 counts/min. 



"^ Atom per cent excess for N'* and C", counts/min. as BaCOs at infinitive thickness for C'"'. 



■^ Calculated by difference. 



were carried out with L-aspartic acid, DL-ureidosuccinic acid, and L-glu- 

 tamine. Tiie results are summarized in Tables XI and XII. 



In this system ammonia-N^^ was incorporated equally into positions 1 

 and 3. The presence of glutamine did not affect this distribution. Aspartic 

 acid, however, under proper conditions, diluted the isotope content of posi- 

 tion 3. This, together with the finding that in a shorter experiment the N^^ 

 from aspartic acid showed up mainly in position 3, makes the amino group 

 of aspartic acid a precursor of nitrogen 3. Unlabeled DL-ureidosuccinic 

 acid considerably diluted the N'^ incorporation of ammonia into orotic 

 acid. Furthermore, when carrier ureidosuccinic acid was added after in- 

 cubation in the same experiment, it was found that the ureidosuccinic acid 



