126 PTEROYLGLUTAMIC ACID 



on thymine was tested by autoclaving the cells with dilute hydrochloric 

 acid and assaying with L. casei. A "plateauing" of the L. casei assay response 

 at approximately half -maximum growth was observed. This response is 

 characteristic of that obtained with thymine and suggests that the material 

 in the S. faecalis R cells giving the response is thymine and not PGA. 

 These facts led to the suggestion that PGA functioned directly or indirectly 

 as a coenzyme in the synthesis of thymine by S. faecalis R. 



Hitchings et al} studied the effect of a large number of pyrimidines on the 

 growth of L. casei and found that only those compounds which retain a 

 methyl group in the 5 position possess any activity. Substitution of an 

 imino group for one oxygen such as in 5-methylcytosine or 5-methylisocyto- 

 sine yields compounds one-tenth as active as thymine. Replacement of 

 both oxygens by imino groups as in 5-methyl-2 , 4-diaminopurine results in 

 a still further decrease of activity. Replacement of the 5-methyl group by 

 oxygen, amino groups, or halides produces inhibitory compounds. Of 

 special interest is 5-bromouracil, which inhibits completely the growth of 

 L. casei with thymine as the nutrient but which produces slight growth 

 stimulation when a "folic acid concentrate" (PGA) is used as a nutrient. 



Further evidence concerning the function of PGA in purine and pyrimi- 

 dine synthesis has been provided by the work of Rogers and Shive^ using 

 the method of inhibition analysis. The antagonist employed was "x methyl 

 PGA," which was prepared from Q:,|Q-dibromobutyraldehyde, and which 

 had been shown by Franklin et al.^ to function competitively as a PGA 

 antagonist for rats and microorganisms. The inhibition ratios (ratio of 

 inhibitor to PGA for complete inhibition) for this antagonist ^^ere deter- 

 mined in the presence of various purines and thymine. Thymine was found 

 to have no effect on the toxicity of "x methyl PGA" in the absence of a 

 purine but did counteract it when a purine was present. The inhibition ratio 

 in the absence of a purine was approximately 30. The addition of 10 7 of 

 hypoxanthine per milliliter increased it to 100. The further addition of 3 7 

 of thymine per milliliter further increased the antibacterial ratio to about 

 1000. These results also show that the PGA requirement for purine synthe- 

 sis is larger than that for thymine synthesis, since the synthesis of purines 

 is blocked at a lower concentration of antagonist. Thus at certain critical 

 levels of antagonist it is possible effectually to block one enzyme function 

 of a vitamin without seriously impairing another. Inhibition of growth by 

 high levels of the antagonist in the presence of adenine and thymine shows 

 that PGA has still another function. This is to be expected, since with L. 

 casei a combination of thymine plus purine gives only half-maximum 



" G. H. Hitchings, E. A. Falco, and M. B. Sherwood, Science 102, 251 (1945). 

 6 L. L. Rogers und W. Shive, J. Biol. Chem. 172, 751 (1948). 



6 A. L. Franklin, E. L. R. Stokstad, M. Belt, and T. H. Jukes, ./. Biol. Chcm. 169, 

 427 (1947). 



