Abstract
Three different dihydrofolate reductases (I, IIa and IIb) were separated from cell-free extracts of the trypanosomid flagellate Crithidia fasciculata ATCC12857. The major reductase (IIa) was purified 2744-fold by column chromatographies on DEAE-Sephadex, CM-Sephadex, Sephadex G- 150 and folate-Sepharose 4B. The final preparation was homogeneous in electrophoretic analysis. Its molecular weight was estimated by gel filtration to be 110, 000 and consisted of two subunits with the same molecular weight of 58, 000. The optimum pH was 7.0. The enzyme activity depended on dihydrofolate and NADPH. Other folate derivatives, unconjugated pteridines and NADH were ineffective. Km values for dihydrofolate and NADPH were 1.1 and 2.7 μm, respectively. One mol of the enzyme reduced 755 mol of dihydrofolate per min at 30°C. The enzyme activity was inhibited by p-chloromercuribenzoate, N-ethylmaleimide and urea. It was also inhibited by anti-folates, such as methotrexate, aminopterin, pyrimethamine and trimethoprim, with I50 values (concentrations required for 50% inhibition) of 1.4, 1.5, 6400 and 27, 000 nM, respectively.
