The effect of methoirexate on dihydrofolic reductase in the rat liver

Abstract

Inhibition of dihydrofolic reductase in the rat liver by the folic acid antagonist methotrexate, amethopterin, is followed by a rapid recovery of enzyme activity. During serial administration of the antimetabolite, dihydrofolic reductase - methotrexate complexes were shown to be stable to dissociation, and methotrexate was readily accessible at all times to the enzyme. Antifolate treatment induced the synthesis of a dihydrofolic reductase that was present in small amounts in the livers of untreated rats. Two reductases, which were present in approximately equal amounts in some liver extracts from methotrexate-treated rats, were purified by a combination of ammonium sulphate fractionation and chromatography on Sephadex G-100. The molecular weights of the enzymes were estimated to be 20,000 - 500 and 24,500 - 500 respectively for the main reductase component present in the normal rat liver and for the enzyme synthesised in the presence of methotrexate. The two dihydrofolic reductase enzymes migrated in opposite directions when subjected to electrophoresis on cellulose acetate strips at pH 8.5, but were shown to have similar Michaelis constants for dihydrofolate at pH 7.4. pH curves had similar profiles for the two enzymes. Evidence was found that the synthesis of dihydrofolic reductase in the presence of methotrexate involved an enzyme which exhibited some degree of reversibility in its binding of the antifolate. The adaptation of the liver in relation to mechanisms for drug resistance during methotrexate therapy is discussed.