Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Abstract

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology 14: 513-520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5-8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent Kmfor dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent Kmfor dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with Kisof 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with Kisof 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with Kiiof 103 and 23 μM, respectively. © 1984.