ANALOGS OF PTEROYLGLUTAMATE (FOLATE) 585 



might inhibit directly the reactions in which tetrahydrofolate functions. 

 There is essentially no information on this second possibility. Cyclohydro- 

 lase is inhibited weakly by amethopterin (43% at 0.5 mM) and aminopterin 

 (69% at 0.5 mM) (Tabor and Wyngarden, 1959), and formyltetrahydro- 

 folate synthetase is even more weakly inhibited by several analogs ( Jaenicke 

 and Erode, 1961; Whiteley et al., 1959). The many other reactions involved 

 have never been examined for inhibition by analogs. All one can say at the 

 present time is that the known inhibitions on folate reduction are sufficient 

 to explain most or all of the effects of these analogs. 



Some examples of the inhibition of syntheses will be mentioned to illus- 

 trate the nature of the metabolic actions of these analogs. Aminopterin and 

 amethopterin invariably depress the incorporation of formate-C^* into pu- 

 rines and nucleic acids; this has been shown in rabbit bone marrow (Totter 

 and Best, 1955), leukemic spleen extracts (Balis and Dancis, 1955), and the 

 whole animal (Skipper et al., 1950). It appears that thymine synthesis is 

 more sensitive than purine synthesis to inhibition by these analogs. In bone 

 marrow 0.0021 mM aminopterin inhibits incorporation into thymine 72% 

 but into adenine or guanine only 22%. It would be interesting to know 

 what the effect on adenine nucleotides is, but little is known. Aminopterin 

 elevates ATP in liver, has no effect on spleen or muscle ATP, and reduces 

 tumor ATP (Zahl and Albaum, 1955). Indeed, the total adenine nucleotides 

 in the sarcoma decrease. One might expect the effects to depend on the 

 relative rates of adenine synthesis and turnover in the tissues. Aminopterin 

 lowers liver NAD levels — 39% fall at 60 //g/day and 56% fall at 100 //g/day 

 — and if this occurs throughout the body it could be an important conse- 

 quence of interference with folate metabolism (Strength et al., 1954). The 

 in vitro depression of respiration by aminopterin is not completely reversed 

 by added NAD, and it is very possible that other coenzymes (e.g. NADP, 

 coenzyme A. or FAD) are decreased. 



The interconversion of glycine and serine is inhibited by aminopterin 

 when only folate is supplied, but the activity is restored with folinate (Blak- 

 ley, 1954). Tetrahydroaminopterin does not inhibit serine synthesis when 

 folinate is provided, but 2-deaminofolate inhibits some 55% at 0.75 mM in 

 rabbit liver extracts (Blakley, 1957). The incorporation of formate-C^^ into 

 lymphoma proteins is inhibited 72% by 0.0073 mM amethopterin, but little 

 effect is observed in normal liver (Williams et al., 1955). In general these 

 analogs block nucleic acid synthesis more than protein synthesis, but this 

 may vary a good deal from one tissue to another, or one organism to an- 

 other. Most of the inhibitions in nucleic acid and protein synthesis have been 

 attributed solely to defects in the formation of the constituent units, and 

 little consideration has been given to other possible contributing factors, 

 such as lowered levels of various coenzjTnes with impairment of oxidative 

 and phosphorylative reactions. 



