The multiple roles of the mitochondrion of the malarial parasite.

Mitochondria of the malaria parasite Plasmodium falciparum are morphologically different between the asexual and sexual blood stages (gametocytes). In this paper recent findings of mitochondrial heterogeneity are reviewed based on their ultrastructural characteristics, metabolic activities and the differential expression of their genes in these 2 blood stages of the parasite. The existence of NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV) suggests that the biochemically active electron transport system operates in this parasite. There is also an alternative electron transport branch pathway, including an anaerobic function of complex II. One of the functional roles of the mitochondrion in the parasite is the coordination of pyrimidine biosynthesis, the electron transport system and oxygen utilization via dihydroorotate dehydrogenase and coenzyme Q. Complete sets of genes encoding enzymes of the tricarboxylic acid cycle and the ATP synthase complex are predicted from P. falciparum genomics information. Other metabolic roles of this organelle include membrane potential maintenance, haem and coenzyme Q biosynthesis, and oxidative phosphorylation. Furthermore, the mitochondrion may be a chemotherapeutic target for antimalarial drug development. The antimalarial drug atovaquone targets the mitochondrion.

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.

Characterisation of carbonic anhydrase in Plasmodium falciparum.

Here we report the existence, purification and characterisation of carbonic anhydrase in Plasmodium falciparum. The infected red cells contained carbonic anhydrase approximately 2 times higher than those of normal red cells. The three developmental forms of the asexual stages, ring, trophozoite and schizont were isolated from their host red cells and found to have stage-dependent activity of the carbonic anhydrase. The enzyme was purified to homogeneity from the crude extract of P. falciparum using multiple steps of fast liquid chromatographic techniques. It had a Mr of 32 kDa and was active in a monomeric form. The human red cell enzyme was also purified for comparison with the parasite enzyme. The parasite enzyme activity was sensitive to well-known sulfonamide-based inhibitors of both bacterial and mammalian enzymes, sulfanilamide and acetazolamide. The kinetic properties and the amino terminal sequences of the purified enzymes from the parasite and host red cell were found to be different, indicating that the purified protein most likely exhibited the P. falciparum carbonic anhydrase activity. In addition, the enzyme inhibitors had antimalarial effect against in vitro growth of P. falciparum. Moreover, the vital contribution of the carbonic anhydrase to the parasite survival makes the enzyme an attractive target for therapeutic evaluation.

Ultrastructure and function of mitochondria in gametocytic stage of Plasmodium falciparum.

Morphological properties of the mitochondrial organelles in the asexual and sexual gametocytic stages of Plasmodium falciparum have been analyzed and found to be markedly different. From in vitro cultures of both stages in human erythrocytes, it has been demonstrated that the asexual stages contained a defined double-membrane organelle having a few tubular-like cristae. The numbers of mitochondria in the gametocytes were found to be approximately 6 organelles per parasite, and they showed a greater density of the cristae than that of the asexual stage parasite. The organelles of the gametocytes were successfully purified by differential centrifugation following Percoll density gradient separation with the results of approximately 7% yields and approximately 5 folds. The gametocytic organelles contained much more activities of mitochondrial electron transporting enzymes (i.e., cytochrome c reductase, cytochrome c oxidase) than the asexual stage organelles. Mitochondrial function as measured by oxygen consumption were found to be different between these two stages organelles. Their rates of oxygen consumption were relatively low, as compared to those of human leukocyte and mouse liver mitochondria. In contrast to the coupled mammalian mitochondria, the gametocytic organelles were in the uncoupling state between oxidation and phosphorylation reactions during their respiration. However, they were sensitive to inhibitors of the electron transport system, e.g., antimycin A, cyanide. Our results suggest that the mitochondria of the gametocytic stages are metabolically active and still underdeveloped, although their inner membranes are extensively folded. The biochemical significance of the unique structure of the mitochondria in these developing stages in host erythrocytes remains to be elucidated.

Purification and characterization of Plasmodium falciparum succinate dehydrogenase.

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 Km values of 3 and 0.2 microM, 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 (approximately 600-5000 M(-1) s(-1)). Fumarate, malonate and oxaloacetate were found to inhibit the malarial enzyme with Ki values of 81, 13 and 12 microM, 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 microM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 microM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 microM. 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 microM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme.

Molecular characterization of mitochondria in asexual and sexual blood stages of Plasmodium falciparum.

Molecular mechanisms that regulate gene expression during development of asexual stage to sexual stage of Plasmodium falciparum in the human erythrocyte are largely unknown. There were apparent variations in ultrastructural characteristics of the mitochondrion between the two developing stages. The asexual stage's mitochondrion had developed less than that of the sexual stage. The respiratory complexes of the mitochondrial electron transport system in the asexual stage were approximately 8-10 times less active than those in the sexual stage. Using quantitative polymerase chain reaction to amplify the cytochrome b gene encoding a subunit of mitochondrial cytochrome c reductase, the amount of the cytochrome b gene of the sexual stage was calculated to be approximately 3 times higher than that obtained from the asexual stage. Moreover, using quantitative reverse-transcription polymerase chain reaction, a relatively high level of approximately 1.3-kb transcript mRNA of the cytochrome b gene was observed in the sexual stage compared to the asexual stage. A known single-copy chromosomal dihydrofolate reductase gene was found to have a similar amount in the two stages. These results suggest that the copy number of the mitochondrial gene, including transcriptional and translational mechanisms, plays a major regulatory role in differential expression during the development of the asexual to sexual stage of P. falciparum in the human cell.

Mitochondrial oxygen consumption in asexual and sexual blood stages of the human malarial parasite, Plasmodium falciparum.

The two developmental stages of human malarial parasite Plasmodium falciparum, asexual and sexual blood stages, were continuously cultivated in vitro. Both asexual and sexual stages of the parasites were assayed for mitochondrial oxygen consumption by using a polarographic assay. The rate of oxygen consumption by both stages was found to be relatively low, and was not much different. Furthermore, the mitochondrial oxygen consumption by both stages was inhibited to various degrees by mammalian mitochondrial inhibitors that targeted each component of complexes I- IV of the respiratory system. The oxygen consumption by both stages was also affected by 5-fluoroorotate, a known inhibitor of enzyme dihydroorotate dehydrogenase of the pyrimidine pathway and by an antimalarial drug atovaquone that acted specifically on mitochondrial complex III of the parasite. Moreover, antimalarials primaquine and artemisinin had inhibitory effects on the oxygen consumption by both stages of the parasites. Our results suggest that P. falciparum in both developmental stages have functional mitochondria that operate a classical electron transport system, containing complexes I-IV, and linked to the pyrimidine biosynthetic pathway.

Antimalarials from Stephania venosa, Prismatomeris sessiliflora, Diospyros montana and Murraya siamensis.

Fourteen compounds isolated from Stephania venosa, Prismatomeris sessiliflora, Diospyros montana and Murraya siamensis were tested for their antimalarial potential. The 6a,7-dehydroaporphine alkaloids dehydrostephanine and dehydrocrebanine showed potent activity with IC50 values of 40 and 70 ng/ml, respectively. The 13C-NMR data of rubiadin, rubiadin-1-methyl ether, diospyrin and 5-hydroxy-4-methoxy-2-naphthal-dehyde were extensively studied.

Antimalarial naphthoquinones from Nepenthes thorelii.

Roots of Nepenthes thorelii yielded plumbagin, 2-methylnaphthazarin, octadecyl caffeate, isoshinanolone, and droserone. In addition, seven derivatives were prepared from plumbagin. Each of these natural and semisynthetic compounds was evaluated for in vitro antimalarial potential.

Xanthones with antimalarial activity from Garcinia dulcis.

Chromatographic separation of the EtOH extract of the bark of Garcinia dulcis (Guttiferae) furnished five xanthones, viz 1,7-dihydroxyxanthone (1), 12b-hydroxy-des-D-garcigerrin A (2), 1-O-methylsymphoxanthone (3), symphoxanthone (4), and garciniaxanthone (5). These xanthones 1-5 showed inhibitory effects on the growth of Plasmodium falciparum with IC50 values of 0.96-3.88 micrograms/ml. In addition, revised 13C-NMR assignments of 3 and complete 13C-NMR assignments of 4 were obtained through analysis of their COSY, NOESY, HMQC, and HMBC spectra.

Antimalarial xanthones from Garcinia cowa.

Five xanthones from the bark of Garcinia cowa, namely 7-O-methylgarcinone E (1), cowanin (2), cowanol (3), cowaxanthone (4), and beta-mangostin (5), were found to possess in vitro antimalarial activity against Plasmodium falciparum with IC50 values ranging from 1.50 to 3.00 micrograms/ml. Complete 1H- and 13C-NMR assignments of these compounds are also reported.

Mitochondrial ubiquinol-cytochrome c reductase and cytochrome c oxidase: chemotherapeutic targets in malarial parasites.

In order to demonstrate that the mitochondrial electron transport system may be a target for antimalarial drug design in the human malarial parasite Plasmodium falciparum, ubiquinol-cytochrome c reductase and cytochrome c oxidase were purified from mitochondria of the parasite cultivated in vitro. It was found that the catalytic efficiency of the two enzymes from the malarial parasite were markedly lower than those from mouse liver mitochondria. The classical inhibitors affecting different quinone binding sites of the mammalian reductase, antimycin and myxothiazole, which had little antimalarial activities on P.falciparum growth in vitro, were found to exhibit little inhibitory effect against the parasite reductase. The malarial parasite reductase was more sensitive to inhibition by the antimalarial drug, 2-[trans-4-(4'-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, than the mammalian enzyme, suggesting both the therapeutic potential of the target and the drug.

Mitochondrial cytochrome b gene in two developmental stages of human malarial parasite Plasmodium falciparum.

The cytochrome b gene of the mitochondrial ubiquinol-cytochrome c reductase (complex III of electron transport chain) was characterized in two developmental stages of human malarial parasite cultivated in vitro. The cytochrome b gene spanning the nucleotide position 4691 to 5930 in 6-kb mitochondrial DNA from gametocytic (sexual) and intraerythrocytic (asexual) stages of Plasmodium falciparum (a T9,94 mutant line) were in vitro amplified from total DNA using polymerase chain reaction (PCR). It was found that the parasites from both stages contained the PCR product approximately 1.2 kb in length that was localized in mitochondria. The nucleotide sequences of cytochrome b gene at Qi/quinone binding site from both stages were analyzed using thermal cycle sequencing and were found to be the same. The amount of this gene from both stages of the parasite were determined by using the quantitative PCR method. The results showed that the amount of the cytochrome b gene produced from the sexual stage was seven times higher than that obtained from the asexual stage. Our results would provide basic information on the regulation of cytochrome b and the 6-kb mitochondrial DNA during growth and development of the sexual and asexual stages of the malarial parasite in the mammalian host.

Purification, characterization and localization of mitochondrial dihydroorotate dehydrogenase in Plasmodium falciparum, human malaria parasite.

The mitochondrial dihydroorotate dehydrogenase (DHODase), the single redox reaction in the pyrimidine de novo synthetic pathway, was purified to near homogeneity by detergent solubilization and fast protein liquid chromatography (FPLC) techniques from the mature trophozoites and schizonts of Plasmodium falciparum, human malaria parasite. The purified DHODase was monofunctional protein with a M(r) of 56,000 +/- 4000, based on Superose 12 gel filtration FPLC and SDS-PAGE analyses. Polyclonal antibodies raised against the purified P. falciparum protein was cross-reacted with P. berghei, rodent malaria parasite. The optimal activity of DHODase required long chain of coenzyme Q (CoQ6-10) which were essential for electron transfer. The Km and kcat values for L-dihydroorotate were 14.4 +/- 5.9 microM and 15.0 +/- 1.4 min-1, respectively; for CoQ6, they were 22.5 +/- 6.4 microM and 21.6 +/- 3.4 min-1. L-Orotate, an enzymatic product, was a strong competitive inhibitor with Ki of 18.2 +/- 3.6 microM. The 5-substituted L-orotates having antimalarial activities against P. falciparum in vitro were found to be competitive inhibitors. The inhibitory effect by these 5-substituted L-orotates on the malarial DHODase was different from the mammalian enzyme. Various benzoquinones and naphthoquinones were found to inhibit the purified DHODase activity at a different degree. Mitochondria from erythrocytic cycle of P. falciparum were purified, using differential centrifugation and followed by Percoll density gradient separation, with purifications of 13-fold and overall yields of 33%. The double-membraned mitochondria had a few tubular-like cristae structure as what found in many protozoan parasites. DHODase was localized inside the mitochondria as probed by immunogold labeling with the polyclonal antibodies and selective solubilization by digitonin.

Plasmodium berghei: partial purification and characterization of the mitochondrial cytochrome c oxidase.

Mitochondria from a rodent malarial parasite (Plasmodium berghei) were successfully purified by differential centrifugation and 22% Percoll density gradient separation. The purified mitochondria from the erythrocytic stages of the parasite had a density of 1.05 and were found to be heterogeneous by transmission electron microscopy and rhodamine 123 fluorescence microscopy. Three marker enzymes, dihydroorotate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase, were assessed during the organelle separation. Purification of cytochrome c oxidase was carried out from the purified mitochondria by using combination techniques of detergent solubilization and reduced cytochrome c-agarose affinity chromatography. The 560-fold purified enzyme with 3.6% yield was obtained and it had low catalytic efficiency with a kcat/Km of 5.9 x 10(-5) M-1 x min-1. The native form of the enzyme, determined by a gel filtration column on fast protein liquid chromatography, was found to be an oligomeric structure with a minimal molecular weight of 670 kDa. The malarial enzyme was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then compared to the enzyme obtained from host liver cells. These results suggested that the partially purified enzyme from the parasite was not different from its host mammalian cells. The importance of the enzyme in the erythrocytic phase of the parasite is discussed as a part of a simple electron transport system in mitochondrion linked to limited oxygen utilization and pyrimidine de novo biosynthesis.

Antimalarial activity of orotate analogs that inhibit dihydroorotase and dihydroorotate dehydrogenase.

Dihydroorotase and dihydroorotate dehydrogenase, two enzymes of the pyrimidine biosynthetic pathway, were purified from Plasmodium berghei to apparent homogeneity. Orotate and a series of 5-substituted derivatives were found to inhibit competitively the purified enzymes from the malaria parasite. The order of effectiveness as inhibitors on pyrimidine ring cleavage reaction for dihydroorotase was 5-fluoro orotate greater than 5-amino orotate, 5-methyl orotate greater than orotate greater than 5-bromo orotate greater than 5-iodo orotate with Ki values of 65, 142, 166, 860, 2200 and greater than 3500 microM, respectively. 5-Fluoro orotate and orotate were the most effective inhibitors for dihydroorotate dehydrogenase. In vitro, 5-fluoro orotate and 5-amino orotate caused 50% inhibition of the growth of P. falciparum at concentrations of 10 nM and 1 microM, respectively. In mice infected with P. berghei, these two orotate analogs at a dose of 25 mg/kg body weight eliminated parasitemia after a 4-day treatment, an effect comparable to that of the same dose of chloroquine. The infected mice treated with 5-fluoro orotate at a lower dose of 2.5 mg/kg had a 95% reduction in parasitemia. The effects of the more potent compounds tested in combination with inhibitors of other enzymes of this pathway on P. falciparum in vitro and P. berghei in vivo are currently under investigation. These results suggest that the pyrimidine biosynthetic pathway in the malarial parasite may be a target for the design of antimalarial drugs.

Malarial dihydroorotate dehydrogenase mediates superoxide radical production.

Dihydroorotate dehydrogenase purified from mitochondria of Plasmodium berghei, a rodent malaria parasite, mediates production of superoxide radical during oxidation of dihydroorotate to orotate. Reduction of dichlorophenolindophenol or cytochrome c or nitroblue tetrazolium was significantly inhibited by superoxide dismutase or theonyltrifluoroacetone, a specific iron chelator of the enzyme. These results, together with the recent evidence of manganese-superoxide dismutase activity in malarial mitochondria [Ranz, A., and Meshnick, S.R. (1989) Exp. Parasitol. 69, 125-128], suggest that the production of superoxide radical may occur in vivo.

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Folate and cobalamin metabolism in Plasmodium falciparum.

During the past several years, Jerapan Krungkrai, H. Kyle Webster and Yongyuth Yuthavong have characterized the metabolic pathway of folate biosynthesis and folate-dependent reactions, including the cobalamin-dependent activity of methionine synthase, in P. falciparum grown in vitro. In this review, they discuss the implications of this work for understanding the mechanism of pyrimethamine resistance and the importance of cloning the dihydrofolate reductase gene. In addition, the role of cobalamin in P. falciparum will be considered. Interference with cobalamin use may represent a new target for combating the parasite.

Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata.

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamoyl-L-aspartate (L-CA) to L-5,6-dihydroorotate (L-DHO), which is the third enzyme in de novo pyrimidine biosynthesis. The enzyme was purified from two parasitic protozoa, Crithidia fasciculata (about 16,000-fold) and Plasmodium berghei (about 790-fold). The C. fasciculata enzyme had a native molecular weight (Mr) of 42,000 +/- 5000, determined by gel filtration chromatography, and showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Mr 44,000 +/- 3000. The DHOase from P. berghei had a native molecular weight of 40,000 +/- 4000 and a subunit molecular weight on SDS-PAGE of 38,000 +/- 3000. The DHOase from both parasites, in contrast to the mammalian enzyme which resides on a trifunctional protein of the first two enzymes of the pathway, carbamoyl-phosphate synthase and aspartate transcarbamylase, is monomeric and has no oligomeric structure as studied by chemical cross-linking with dimethyl suberimidate. The rate of cyclization of L-CA by the C. fasciculata enzyme was relatively high at acidic pH, decreasing to a very low rate at alkaline pH. In contrast, the rate of ring cleavage of L-DHO was very low at acidic pH and increased to a higher rate at alkaline pH. These pH-activity profiles gave an intersection at pH 6.6. The Km and kcat for L-CA were 0.846 +/- 0.017 mM and 39.2 +/- 6.4 min-1, respectively; for L-DHO, they were 25.85 +/- 2.67 microM and 258.6 +/- 28.5 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)