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In a number of mammalian species, including man, the 4-amino analogs of pteroylglutamate persist in liver and kidney for many months after administration (1-4). In the rat (3) and mouse (4), the binding site appears to be the enzyme dihydrofolate reductase [also termed folate reductase (5) and tetrahydrofolate dehydrogenase.1] Studies with partially purified dihydrofolate reductase obtained from several sources (6-8) have shown the affinity of this enzyme for the 4-amino analogs to be extremely high.Recent work with tritium-labeled pteroylglutamate in vivo has shown that, in man, the labeled compound can be displaced from cells several days after its administration, both by unlabeled pteroylglutamate (9, 10) and by other compounds that possess an affinity for the enzyme dihydrofolate reductase (11). If, as animal experiments suggest, the 4-amino analogs bind to the same enzyme, a similar procedure should also bring about the displacement of these compounds. The studies reported here show that, in man, tritiated methotrexate (amethopterin; 4-amino-AN10-methylpteroylglutamic acid) can be readily displaced

A study of the phenomenon of “induction” of the enzyme dihydrofolate reductase that occurs in leukocytes and erythrocytes after methotrexate administration has been made with the dog as an experimental model as weU as man. The “induction” has been shown to be specific for 4‐amino analogues of folate that are strong inhibitors of dihydrofolate reductase. Attempts to block induction with possible end products of dihydrofolate reductase activity and an inhibitor of protein synthesis, actinomycin D, were unsuccessful. Based upon studies of the enzymeinhibitor interaction as a function of pH, the rise in enzyme activity observed is explained by an accumulation of methotrexate‐bound enzyme that occurs as a result of protection of the enzyme from normal catabolic processes; free enzyme is observed because of the in vitro conditions employed for enzyme assay.

The synthesis of 5-methyl-5,6,7,8-tetrahydropteroly tri-, pena-, and heptaglutamate has been accomplished by reductive methylation of the tetrahydropteroyl oligoglutamate with formaldehyde, followed by purification on DEAE-Sephadex. The corresponding [5-14-C]methyltetrahydropteroyl oligoglutamates were prepared from 14-CH-2-0, and tested as substrates for methionine synthetase (EC 2.1.1.13) ISOLATED FROM BOVINE BRAIN. In all cases, the polyglutamate conjugates were better substrates (lower Km, higher Vmax) than the corresponding monoglutamate forms. In addition, the nonradioactive methyltetrahydropteroyl oligoglutamates inhibited the methylation of homocysteine by methyltetrahydrofolate. This indicates that the monoglutamate and polyglutamates compete for the same enzyme, and established a role for the ubiquitous methyltetrahydropteroyl oligoglutamates in mammalian methionine biosynthesis.