476 R. E. HANDSCHUMACHER AND A. D. WELCH 



same extent as with extracts obtained from the parent line, while intact 

 resistant cells in vitro were unaffected by the same concentrations of the 

 antagonist. 152 Similar results have been obtained with a subline of TA3 

 carcinoma 151 ; however, in this case, the resistant cells which were exposed 

 to azaserine in vivo recovered over 50 % of their normal ability to synthesize 

 purines after 24 hours, at a time when these reactions in the cells of the 

 sensitive line were still inhibited by about 85%. 



Although the primary site of action of azaserine and DON appears to be 

 the enzyme concerned with the donation of the amino group of glutamine 

 to formylglycinamide ribonucleotide, several other sites of action have been 

 demonstrated. Inhibition of the initial step in the synthesis of purine ribo- 

 nucleotides, i.e., the formation of ribosylamine-5-phosphate from glutamine 

 and pyrophosphorylribose-5-phosphate, requires approximately fifty times 

 the level of azaserine which is necessary to inhibit the enzyme discussed 

 above. 141 In this initial reaction, the simultaneous addition of glutamine in 

 excess will nullify most of the inhibition caused by azaserine, and DON is 

 a much more effective inhibitor than azaserine. 153 Similar results have been 

 obtained by others, 145 ' 154 and, at even higher levels of azaserine, inhibition 

 of the conversion of xanthylic acid to guanylic acid has been observed. 113 ' 114 



Interference in pyrimidine metabolism by DON and azaserine has been 

 detected only in the conversion of uracil nucleotides to cytidine nucleotides. 

 Thus, DON inhibited the incorporation of carbamyl-L-aspartic and orotic 

 acids into cytosine, as compared to uracil or thymine, in the nucleic acids 

 of tumors and normal tissues of rats in vivo. nb Similar results were obtained 

 with tissue slices, but prevention of the effects of DON by glutamine was 

 not very effective. In whole cell suspensions of the Novikoff hepatoma, 

 DON profoundly inhibited the conversion of orotic acid to cytidine ribo- 

 nucleotides, and these findings were confirmed in studies with a cytoplasmic 

 fraction. 155 



Although the sites of enzymic inhibition established by either azaserine 

 or DON have been clearly indicated as those which utilize glutamine, the 

 mechanisms by which apparently unrelated amino acids, such as phenyl- 

 alanine and tryptophan, prevent inhibition of growth by azaserine in mi- 

 crobial systems, have yet to be explained. 140156 It may be that these com- 

 pounds nullify the action of azaserine by preventing access to receptor sites 



152 E. P. Anderson, B. Levenberg, and L. W. Law, Federation Proc. 16, 145 (1957). 



163 \y. Barg, E. Boggliano, N. Sloane, and E. C. DeRenzo, Federation Proc. 16, 150 

 (1957). 



164 J. S. Gots and E. G. Gollub, J. Bacleriol. 72, 858 (1956). 



165 H. O. Kammen and R. B. Hurlbert, Cancer Research 19, 654 (1959). 



156 H. E. Skipper and J. R. Thomson, in "Ciba Foundation Symposium on Amino 

 Acids and Peptides with Antimetabolite and Cytotoxic Action," (G. E. W. Wol- 

 stonholme and C. M. O'Connor, eds.), p. 38. Churchill, London, 1958. 



