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



lizes F extremely poorly as compared to a derivative of FH 4 , folinic 

 acid (/ 5 FH 4 ), 25 can be inhibited by relatively high concentrations of amethop- 

 terin; however, when substrains of the organism were selected for their 

 capacity to grow on relatively minute amounts of folic acid, the sensitivity 

 to amethopterin was markedly increased (over sixtyfold) ; this situation was 

 observed even when the new strain was grown on folinic acid. 26 



Initially, rat liver slices were shown to convert folic acid enzymically to 

 compounds which, in the presence of ascorbic acid (or certain other reduc- 

 ing agents), could be altered (nonenzymically) to folinic acid 27 ; the enzymic 

 phase of these alterations of folic acid was markedly inhibited by aminop- 

 terin. 28 It has been concluded 12 from studies with avian liver systems that 

 the principal site of action of the 4-amino analogs of F is a TPNH-depend- 

 ent, dihydrofolic (FH 2 ) (7,8-dihydropteroylglutamic) acid reductase which 

 catalyzes the reaction: FH 2 + TPNH + H+ ^ FH 4 + TPN+ (an equili- 

 brium which at neutral reaction lies far to the right) ; in addition, the con- 

 version of F to FH 2 was reported to be TPNH-dependent. However, very 

 recent studies 29 have indicated that although the conversion of folic acid 

 to FH 2 is TPNH-dependent, that of FH 2 to FH 4 (with extracts of sheep 

 liver at pH 5) requires DPNH. In this system the reduction of F to FH 2 

 was exceedingly sensitive to amethopterin; in fact, the enzyme could be 

 titrated with the inhibitor, 1.2 X 10~ 6 /xmole being equivalent to 

 that amount of enzyme which, under prescribed conditions, reduced 1 X 

 10 -3 Mmole of folic acid in 30 minutes. Under these conditions, the affinity 

 of the enzyme for aminopterin was almost 1,000 times that for folic acid. 

 Earlier evidence also showed that the enzymic reduction of F to FH 2 (by 

 chicken liver) is inhibited by the 4-amino antagonists 30 ; and that the over- 

 all system of chicken liver (F to FH 4 ) is dependent on TPNH and is 

 exceedingly sensitive to aminopterin 31 or amethopterin. 32 In the former 

 study, 31 the capacity of DPNH to substitute for TPNH in the reduction 

 of FH 2 was demonstrated. Recent studies 33 have demonstrated firm binding 

 of aminopterin or amethopterin in the supernatant fraction of a sucrose 

 homogenate of rat liver prepared 16 hours after the injection of a large dose 



25 H. E. Sauberlich and C. A. Baumann, J. Biol. Chem. 176, 165 (1948); 181, 871 

 (1949). 



26 C. A. Nichol, Nature 183, 550 (1959). 



27 C. A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 52 (1950). 



28 C. A. Nichol and A. D. Welch, Proc. Soc. Exptl. Biol. Med. 74, 403 (1950). 



29 J. M. Peters and D. M. Greenberg, Nature 181, 1669 (1958); Biochim. et Biophys. 

 Acta 32, 273 (1959). 



30 S. Futterman, J. Biol. Chem. 228, 1031 (1957). 



31 S. Futterman and M. Silverman, «7\ Biol. Chem. 224, 31 (1957). 



32 S. F. Zakrzewski and C. A. Nichol, Biochim. et Biophys. Acta 27, 427 (1958). 



33 W. C. Werkheiser, Proc. Am. Assoc. Cancer Research 3, 72 (1959). 



