Specific Binding of 4-Amino Folk Acid Analogues by Folic Acid Reductase*

The early studies of Fountain et al. (I) demonstrated clearly that injected amethopterin’ persists in certain mouse tissues such as liver and kidney for periods up to 8 months. The suggestion was made by these authors and by others (2,3) that this retention was due to binding of the drug to an enzyme, perhaps one involved in the conversion of folate to tetrahydrofolate. Such prolonged retention implies an exceptionally tight binding which might be observable in vitro. This paper describes experiments which demonstrate that the agent which binds amethopterin and related 4-amino derivatives of folic acid is the enzyme folic acid reductase and that this binding is specific and exceedingly tight. A preliminary report of a portion of this work has appeared elsewhere (4).