Plasmodium berghei Dihydrofolate Reductase Isolation, Properties, and Inhibition by Antifolates

Dihydrofolate reductase has been isolated from cells of the rodent malarial organism Plasmodium berghei by separation from the host cells with saponin and extraction by rupture in a French pressure cell. The enzyme exhibits certain distinctive properties which clearly distinguish it from the dihydrofolate (H 2 -folate) reductase isolated from the host cells (mouse erythrocytes) and from H 2 -folate reductases obtained from other sources, as reported in the literature. The molecular weight determined by gel filtration (190,000 ± 10%) is 9-10-fold higher than reported for most other H 2 -folate reductases. This value was not significantly decreased when the enzyme was passed over Sephadex G-100 columns in the presence of KCl, urea + 2-mercaptoethanol, or the substrate, H 2 -folate. The plasmodial H 2 -folate reductase is unlike bacterial enzymes in its stimulation by high concentrations of KCl and urea, and its approximately 10-fold lower K m value for NADPH (1.3 µM). The enzyme from P. berghei differs markedly from other dihydrofolate reductases in its sensitivity to several diaminoheterocyclic inhibitors. Most striking is the inhibition by pyrimethamine; it exhibits a 50% inhibitory concentration of approximately 0.5 nM compared with 1 µM for the mouse erythrocyte enzyme. The binding of pyrimethamine to P. berghei dihydrofolate reductase is stoichiometric when enzyme and drug have been incubated for 5-10 min prior to the addition of H 2 -folate, and reversible in the absence of prior incubation. A positive correlation was observed between the binding of pyrimethamine and three dihydrotriazines by the enzyme and the activity of these compounds in vivo against P. berghei infections. These data establish that the selective action of pyrimethamine in malaria is due to the greater sensitivity to this drug of the plasmodial enzyme as compared to the host enzyme and reflect the unique, potent binding of pyrimethamine to plasmodial H 2 -folate reductase.