39. ANTIMETABOLITES AND NUCLEIC ACID METABOLISM 459 



of the antagonist. Extensive dialysis failed to remove the analog unless 

 folic acid was present in a high concentration. A direct correlation was dem- 

 onstrated between the amount of antagonist required to inhibit the folic 

 acid-reductase activity in the supernatant fraction of normal liver and the 

 amount of enzyme present. The affinity of the reductases for the 4-amino 

 analogs is so great that a value for "K/' cannot easily be estimated by 

 classic methods; however, the procedure 34 of titrating the inhibitor with 

 the enzyme may be used, as was done in this case. 



Clearly, the 4-amino analogs do not inactivate the susceptible enzymes, 

 as had been proposed 36 ; rather, the affinity of the enzymes for the antago- 

 nists is so great as to make infeasible the physiological attainment of con- 

 centrations of folic acid sufficient to displace the inhibitor from the enzymes. 

 Of considerable interest will be the relative affinities of the folic acid re- 

 ductases of various animal cells and tissues for the 4-amino analogs, since 

 these might account in part for the considerable variations in the responses 

 of various normal tissues, and those of various types of neoplasms. Great 

 differences between the reductases of different animal species are almost 

 certain to be encountered, since in some species, e.g., the guinea pig, aminop- 

 terin and amethopterin are toxic only in relatively enormous doses. These 

 antagonists do not appear to be inactivated significantly or excreted more 

 rapidly by the guinea pig, and in this species, folic acid is enzymically re- 

 duced by the liver to compounds determinable as folinic acid. 36 Finally, 

 the remarkably greater toxicity of amethopterin in female mice, as com- 

 pared to males, might be related to the relative amounts of the reductases, 

 or their affinities for the antagonist, in the two sexes. 37 



As a result of the profound inhibition of the F and FH 2 reductases by the 

 4-amino derivatives of F, the formation and regeneration of the F-derived 

 coenzyme is blocked, and many biochemical transformations are inhibited 

 or prevented. Although FH 4 may be regarded as the coenzyme (or an es- 

 sential portion of it) in each of these reactions, the exact structure of the 

 derivatives of FH 4 involved in the transformations has not yet been es- 

 tablished to the satisfaction of all concerned. 38 In any case, at least five 

 derivatives of FH 4 are involved in reactions of great biological importance; 

 these are 11 : (1) a formyl (/) derivative, either / 10 FH 4 or / 5 " 10 FH 4 + (i.e., a 

 compound with a =CH — group linking nitrogens 5 and 10), in which the 

 substituent is at the formic acid level of oxidation; (2) a so-called hydroxy- 



34 W. W. Ackermann and V. R. Potter, Proc. Soc. Exptl. Biol. Med. 72, 1 (1949). 

 36 E. M. Greenspan, A. Goldin, and E. B. Schoenbach, Cancer 4, 619 (1951). 



36 D. Wood and C. A. Nichol, unpublished research (1957). 



37 A. Goldin, E. M. Greenspan, B. Goldberg, and E. B. Schoenbach, Cancer 3, S49 

 (1950). 



38 R. L. Blakley, Biochem. J. 65, 331 (1957). 



