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GX Chen, C Mueller, M Wendlinger and JW Zolg
Dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADPH+- oxidoreductase; EC 1.5.1.3) was partially purified by affinity chromatography from three clones of the human malaria parasite Plasmodium falciparum. The three clones were representative of pyrimethamine-sensitive (clone 3D7) and pyrimethamine-resistant (clone HB3 and clone 7G8) parasites with ID50 values of 0.53 nM (3D7), 210 nM (HB3), and 540 nM (7G8), when tested in vitro against the drug. The specific activities of the partially purified DHFR differed by less than a factor of 2 between the sensitive clone 3D7 (442 +/- 39 nmol min- 1 mg-1 protein) and the resistant clones HB3 (634 +/- 25 nmol min-1 mg- 1 protein) and 7G8 (565 +/- 85 nmol min-1 mg-1 protein). The number of catalytic sites in partially purified DHFR from the three clones was similar and ranged from 151 to 194 pmol mg-1 protein. The Km value for NADPH was similar in all three clones (4.5-11.6 microM). The Km value for dihydrofolate was altered 13-fold comparing the sensitive clone 3D7 (3.2 +/- 0.6 microM) with the resistant clone HB3 (42.6 +/- 1.6 microM), with the Km for the resistant clone 7G8 falling in between (11.9 +/- 1.2 microM). The inhibition constants for pyrimethamine increased from 0.19 +/- 0.08 nM (3D7) to 2.0 +/- 0.3 nM (HB3) to 8.9 +/- 0.8 nM (7G8). The inhibition by pyrimethamine of the sensitive clone 3D7 was noncompetitive and competitive for the two other clones. The titration of partially purified DHFR with pyrimethamine revealed a 500- fold increase in the concentration of the drug needed to inhibit the DHFR activity by 50%, when the sensitive clone 3D7 (0.18 +/- 0.02 nM) was compared to the resistant clone 7G8 (95 +/- 16 nM). From the comparison of the specific activities and the catalytic center activities with the Km values for the substrate and the inhibition constants for pyrimethamine, both of which are altered in the resistant clones, we conclude that the molecular mechanism for pyrimethamine resistance in the three clones studied is not based on an overproduction of the DHFR but is due to a decreased affinity to antifolates by a structurally altered enzyme.
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