Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a gamma-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-beta-D-arabinofuranosyladenine-5'-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2',3'-dideoxythymidine-5'-triphosphate (IC(50)>400 microM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase gamma. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC(50)>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).     

Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum

Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a γ-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-β- -arabinofuranosyladenine-5′-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2′,3′-dideoxythymidine-5′-triphosphate (IC₅₀>400 μM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase γ. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC₅₀>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp).