Purification and characterization of Plasmodium falciparum succinate dehydrogenase

• Published in: Molecular and biochemical parasitology 2000-02, Vol.105 (2), p.215-222
• Main Authors: Suraveratum, Nongluk, Krungkrai, Sudaratana R, Leangaramgul, Preecha, Prapunwattana, Phisit, Krungkrai, Jerapan
• Format: Article
• Language: English
• Subjects: Animals; Antimalarial agent; Antimalarials - pharmacology; Chemotherapeutic target; Cytochrome c Group - metabolism; Enzyme Inhibitors - pharmacology; Humans; Inhibitory Concentration 50; Kinetics; Mice; Mitochondria, Liver - enzymology; Mitochondrial electron transport complex; Molecular Sequence Data; Naphthoquinones - pharmacology; Oxidation-Reduction; Oxygen - metabolism; Plasmodium falciparum; Plasmodium falciparum - drug effects; Plasmodium falciparum - enzymology; Plasmodium falciparum - growth & development; Succinate dehydrogenase; Succinate Dehydrogenase - antagonists & inhibitors; Succinate Dehydrogenase - chemistry; Succinate Dehydrogenase - isolation & purification; Succinate Dehydrogenase - metabolism; Succinic Acid - metabolism; Thenoyltrifluoroacetone - pharmacology; Ubiquinone - metabolism
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(99)00180-2

Succinate dehydrogenase (SDH), a Krebs cycle enzyme and complex II of the mitochondrial electron transport system,was purified to near homogeneity from the human malarial parasite Plasmodium falciparum cultivated in vitro by FPLC on Mono Q, Mono S and Superose 6 gel filtration columns. The malarial SDH activity was found to be extremely labile. Based on Superose 6 FPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing-PAGE analyses, it was demonstrated that the malarial enzyme had an apparent native molecular mass of 90±8 kDa and contained two major subunits with molecular masses of 55±6 and 35±4 kDa ( n=8). The enzymatic reaction required both succinate and coenzyme Q (CoQ) for its maximal catalysis with K m values of 3 and 0.2 μM, and k cat values of 0.11 and 0.06 min −1, respectively. Catalytic efficiency of the malarial SDH for both substrates were found to be relatively low (∼600–5000 M −1 s −1). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with K i values of 81, 13 and 12 μM, respectively. The malarial enzyme activity was also inhibited by substrate analog of CoQ, 5-hydroxy-2-methyl-1,4-naphthoquinone, with a 50% inhibitory concentration of 5 μM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 μM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 μM. A known inhibitor of mammalian mitochondrial SDH, 2-thenoyltrifluoroacetone, had no inhibitory effect on both the malarial SDH activity and the oxygen uptake of the parasite at a concentration of 50 μM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.