Studies on the interaction of tritium-labeled aminopterin with dihydrofolic reductase

Abstract

The inhibition of leucocyte dihydrofolic reductase by aminopterin, amethopterin, and dichloroamethopterin was measured at inhibitor concentrations ranging from 10 −6 to 10 −9 M. Tritium-labeled aminopterin was used to prepare the enzyme-inhibitor (E-I) complex of both the leucocyte and chicken liver dihydrofolic reductases. Dialysis of the leucocyte E-I complex resulted in a loss of radioactivity which corresponded to the reappearance of enzyme activity. The chicken liver E-I complex, however, was not dissociated by dialysis. The leucocyte E-I complex was readily dissociated by chromatography on DEAE-cellulose using stepwise desorption with KCl. The reactivated enzyme and the free inhibitor appeared in the 0.05 and 0.15 M fractions, respectively. The chicken liver and leucocyte E-I complexes were readily dissociated by fractionation with ammonium sulfate. A turnover number of 50–100 at pH 8.5 was determined for the chicken liver reductase from data on the amount of inhibitor bound to the enzyme in various ammonium sulfate fractions.