title: Purification of the bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum.
abstract: The bifunctional thymidylate synthase-dihydrofolate reductase complex from the human malaria parasite Plasmodium falciparum has been purified to homogeneity using a sequence of separation steps including phenyl-Superose, gel filtration, dye affinity matrix, hydroxyapatite, and anion exchange chromatography. The specific activity of dihydrofolate reductase increased approximately 24,000-fold from 3.3 units mg-1 protein to 79,000 units mg-1 protein after five successive chromatographic steps with a yield of 31%. Both enzyme activities coeluted as a symmetric peak in highly purified preparations, indicating the existence of a bifunctional enzyme complex in P. falciparum. The apparent molecular weight of the native complex was approximately 120,000 as determined by gel filtration. When individual fractions of the anion exchange column were subject to polyacrylamide electrophoresis under denaturing conditions, the increase in intensity of a single band correlated with the amount of both the thymidylate synthase and dihydrofolate reductase activity. Further purification led to an electrophoretically pure protein (yield 2.6%) with an apparent molecular weight of 67,000, suggesting that the bifunctional enzyme complex from P. falciparum is composed of two subunits of identical size and charge.
authors.affiliation: Department of Molecular Biology, Biomedical Research Institute, Rockville, Maryland 20852.
authors.forename: G X
authors.initials: GX
authors.lastname: Chen
authors.forename: J W
authors.initials: JW
authors.lastname: Zolg
mesh_terms: D000818:Animals; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008727:Methotrexate / Q000378:metabolism; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification*; D013940:Thymidylate Synthase / Q000302:isolation & purification*
chemical_list: D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008727:Methotrexate