306 JOHN M. BUCHANAN 



may donate its nitrogen atom to N-l of hypoxanthine, it does so only in- 

 directly through transamination to form the more direct precursor, aspartic 

 acid. 



To the list of purine precursors must be added ribose phosphate. In his 

 studies on the synthesis of purine compounds in pigeon liver homogenates, 

 Greenberg 21 demonstrated that two radioactive purine compounds, hypox- 

 anthine and inosinic acid, are derived from radioactive formate. By com- 

 parison of the change in specific activity of the two products he concluded 

 that the nucleotide was formed first and then degraded to the base. The 

 synthesis of inosinic acid could be stimulated by the addition of ribose- 

 phosphate to the incubation medium. Ribose-5-phosphate or nucleosides 

 which yielded ribose- 1 -phosphate on phosphorolysis served equally well as 

 stimulatory agents of purine synthesis. 



These experiments of Greenberg were based in part on earlier findings 

 of Krebs and his associates 22 • 23 who found that hypoxanthine is formed de 

 novo in pigeon liver slices from unknown precursors. The synthesis was 

 stimulated by the addition of glutamine and oxalacetic acid to the slices, 

 but at the time of these experiments in 1936 it was not possible to determine 

 the role of these substances in the synthesis. In light of the N 15 experiments 

 described above it is evident that glutamine served as the source of two of 

 the nitrogen atoms directly and that oxalacetate participated in the syn- 

 thesis of the other nitrogen donor, aspartic acid. 



The finding by Edson, Model, and Krebs 22 that hypoxanthine accumu- 

 lated in pigeon liver slices emphasized the relative importance of the re- 

 duced purine compounds and contributed to the later discovery that 

 formate rather than C0 2 was the precursor of the carbon atoms 2 and 8 of 

 the purine ring. While hypoxanthine is oxidized to uric acid in the livers 

 of most animals, this compound accumulates in pigeon liver preparations 

 because of the absence of the enzyme, xanthine oxidase, in this tissue (Vol- 

 ume II, Chapter 23). 



III. Enzymic Reactions of Inosinic Acid Synthesis de Novo 



In general terms the procedure for the detection and isolation of the 

 enzymes and intermediates concerned with the synthesis of the purine 

 nucleotides has been to incubate radioactive precursors with crude extracts 

 of pigeon or chicken liver and then to follow the accumulation of radioac- 

 tive nonpurine intermediates. Separation of these new compounds was 

 usually accomplished by use of ion-exchange or paper chromatographic 

 procedures. A number of the intermediates are heterocyclic amine deriva- 



21 G. R. Greenberg, J. Biol. Chem. 190, 611 (1951). 



22 N. L. Edson, H..A. Krebs, and A. Model, Biochem. J. 30, 1380 (1936). 



23 A. Orstrom, M Orstrom, and H. A. Krebs, Biochem. J. 33, 990 (1939). 



