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 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.

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.

The antimalarial action on Plasmodium falciparum of qinghaosu and artesunate in combination with agents which modulate oxidant stress.

The antimalarial activity of qinghaosu (artemisinine) against Plasmodium falciparum in culture was enhanced by increased oxygen tension. Artesunate, a qinghaosu derivative, showed synergistic effects with miconazole, and with doxorubicin, both of which have been suggested to exert their chemotherapeutic effect through increasing the oxidant stress. In contrast, catalase, dithiothreitol and alpha-tocopherol reduced the effectiveness of qinghaosu in vitro. These results suggest that the action of qinghaosu and artesunate might involve increase in oxidant stress on the infected red cells.