128 PTEROYLGLUTAMIC ACID 



the synthesis of methionine, purine, serine, and thymidine by E. coli. The 

 inhibition ratio for sulfanilamide to 79-aminobenzoic acid is 3000. In the 

 presence of methionine it becomes 10,000; in the presence of methionine 

 and purine it is 30,000; and in the presence of methionine, purine, and serine 

 it becomes 100,000. Further addition of thymine increases it to 200,000. 

 Thymine may be partially replaced by PGA. This indicates that the synthe- 

 sis of methionine, purine, serine, and thymine are mediated directly or 

 indirectly by p-aminobenzoic acid and that the synthesis of each of these 

 end products can be blocked in succession by progressively increasing the 

 amounts of sulfanilamide. In order for any end product to be effective, the 

 other end products, whose production is blocked at lower antagonist con- 

 centrations, must be present in the medium. In the presence of methionine, 

 xanthine, and serine and with 2 mg. of sulfanilamide per miUiliter and 

 approximately 3 017 of p-aminobenzoic acid per milliliter, growth is in- 

 hibited. Growth can be restored at this point by the addition of either 30 

 7 of thymine, 0.3 7 of PGA, or 0.010 7 of p-aminobenzoic acid per miUiliter.^" 

 The fact that such a large amount of PGA is required relative to the p-ami- 

 nobenzoic acid is difficult to explain if one assumes that the former is 

 synthesized from the latter. It is possible, however, that the conversion of 

 preformed PGA to a more active intermediate may be limiting. 



Valine synthesis is the next limiting factor after thymine requirements 

 are met. The growth produced by thymine or PGA reaches the maximum 

 in 72 hours, whereas with p-aminobenzoic acid it is reached in 32 hours. 

 Addition of valine at this point permits early growth, showing that this 

 amino acid becomes the limiting factor after inhibition of thymine synthe- 

 sis.i« 



A similar series of products reverse the toxicity of sulfanilamide for 

 Salmonella typhimurium,^^ and methionine, xanthine, thymine, and valine 

 have been reported to function similarly in sulfonamide reversal in Staphijlo- 

 coccus aureus}'^ 



The relationship of these metabolites to sulfonamide inhibition and the 

 possible precursors are shown in Fig. 8. 



B. MECHANISM OF ACTION 



1. Role of 4-Amino-5-imidazolecarboxamide in Purine Synthesis 



If PGA blocks the synthesis of purine, one might expect an intermediate 

 product to accumulate in a system in which purine synthesis is blocked by 

 a minimum amount of antagonist and in which growth is permitted by the 

 addition of a purine. This has not been demonstrated with lactic acid or- 



'^ K. C. Winkler, P. G. de Haan, and J. van de Langerijt, Antonie van Leeuwenhoek 

 J. Microbiol. Serol. 15, 129 (1949), cited by W. Shive, Vitamins and Hormones 9, 

 75 (1951). 



J 



