Mitochondrial NADH Dehydrogenase from Plasmodium falciparum and Plasmodium berghei

Experimental Parasitology 100, 54–61 (2002) doi:10.1006/expr.2001.4674, available online at http://www.idealibrary.com on

Mitochondrial NADH Dehydrogenase from Plasmodium falciparum and Plasmodium berghei

Jerapan Krungkrai,*,1 Rachanok Kanchanarithisak,* Sudaratana R. Krungkrai,† and Sunant Rochanakij‡ *Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Rama 4 Rd., Bangkok 10330, Thailand; †Unit of Biochemistry, Department of Medical Science, Faculty of Science, Rangsit University, Paholyothin Rd., Patumthani 12000, Thailand; and ‡Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Ramkhamhaeng Rd., Bangkok 10240, Thailand

Krungkrai, J., Kanchanarithisak, R., Krungkrai, S. R. and Rochanakij, S. 2001. Mitochondrial NADH dehydrogenase from Plasmodium falciparum and Plasmodium berghei. Experimental Parasitology 100, 54–61. The mitochondrial electron transport system is necessary for growth and survival of malarial parasites in mammalian host cells. NADH dehydrogenase of respiratory complex I was demonstrated in isolated mitochondrial organelles of the human parasite Plasmodium falciparum and the mouse parasite Plasmodium berghei by using the specific inhibitor rotenone on oxygen consumption and enzyme activity. It was partially purified by two sequential steps of fast protein liquid chromatographic techniques from n-octyl glucoside solubilization of the isolated mitochondria of both parasites. In addition, physical and kinetic properties of the malarial enzymes were compared to the host mouse liver mitochondrial respiratory complex I either as intact or as partially purified forms. The malarial enzyme required both NADH and ubiquinone for maximal catalysis. Furthermore, rotenone and plumbagin (ubiquinone analog) showed strong inhibitory effect against the purified malarial enzymes and had antimalarial activity against in vitro growth of P. falciparum. Some unique properties suggest that the enzyme could be exploited as chemotherapeutic target for drug development, and it may have physiological significance in the mitochondrial metabolism of the parasite. 䉷 2002 Elsevier Science (USA) Index Descriptors and Abbreviations: NADH dehydrogenase; malaria; Plasmodium falciparum; Plasmodium berghei; mitochondrial electron transport complex; antimalarial agent; ADP, adenosine 5⬘diphosphate; CoQ, coenzyme Q, ubiquinone, 2,3-dimethoxy-5-methyl1,4-benzoquinone; CoQ8, ubiquinone-40; CoQ10, ubiquinone-50; ETS,

electron transport system; FPLC, fast protein liquid chromatography, SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

INTRODUCTION

NADH:ubiquinone oxidoreductase (EC 1.6.5.3) or NADH dehydrogenase is a catalytic component of the complex I of the electron transport system (ETS). It transfers electrons from NADH to ubiquinone of the aerobic and energy-generating respiratory chain in eukaryotic mitochondria and several prokaryotes (Hatefi 1985). It is the most complicated among the enzymes of the mitochondrial ETS. It has been isolated and characterized from a number of prokaryotic and eukaryotic sources (Braun et al. 1998; Friedrich and Scheide, 2000; Uneo et al. 1996; Yagi et al. 1998; and references cited therein). Relatively little is known about the enzyme in the genus Plasmodium, the intracellular parasitic protozoan. During the asexual blood stage of the human malarial parasite, Plasmodium falciparum, it grows and matures within red cells of the human host. The absence of cristate structure in mitochondrion at the asexual stage of the parasite is demonstrated and this suggests limited metabolic activity of the organelle (Fry and Beesley 1991; Krungkrai 1995;

1

To whom correspondence should be addressed. Fax: (662)-2524986. E-mail: [email protected] or [email protected]

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0014-4894/02 $35.00 䉷 2002 Elsevier Science (USA) All rights reserved.

NADH DEHYDROGENASE OF MALARIAL PARASITES

Krungkrai et al. 1993; Langreth et al. 1978). Energy requirement during this stage is provided by metabolizing glucose primarily by anaerobic glycolysis (Scheibel 1988; Sherman 1979). It is also suggested that the malaria parasite has a functional mitochondrial ETS and an oxygen-requiring system that is necessary for growth and survival (Ginsburg et al. 1986; Krungkrai 2000; Krungkrai et al. 1991, 1999a, 1999b, 2000; Uyemura et al. 2000). Recently the mitochondrial ETS complex II (succinate dehydrogenase), complex III (ubiquinol-cytochrome c reductase), and complex IV (cytochrome c oxidase) have been purified and characterized from both P. berghei and P. falciparum (Krungkrai et al. 1993, 1997; Suraveratum et al. 2000; Takeo et al. 2000). In this study, the NADH dehydrogenase enzyme is demonstrated and partially purified from a rodent parasite, P. berghei, and a human parasite P. falciparum. Physical and kinetic properties of the purified enzyme are investigated and compared to mouse liver mitochondrial ETS complex I either as intact or as partially purified forms. Some properties observed in the malarial NADH dehydrogenase are different from those in the mammalian mitochondrial enzyme. This will provide that the mitochondrial NADH dehydrogenase of the parasite may be a putative chemotherapeutic target for antimalarial drug design.

MATERIALS AND METHODS Cultivation of malarial parasites and preparation of mitochondria. Human parasite P. falciparum (a pyrimethamine-resistant line) was cultivated by the candle jar method of Trager and Jensen (1976), using a 5% hematocrit of human red cells (type O) suspended in RPMI 1640 medium supplemented with 25 mM N-2-hydroxyethylpiperazine-N ⬘2-ethanesulfonic acid, 32 mM NaHCO3 and 10% fresh human serum (type O). Synchrony of the culture was maintained by the sorbitol lysis procedure of Lambros and Vanderberg (1979). The trophozoite stage of the culture was harvested when parasitemia was ⬃30%. A rodent parasite, P. berghei, was cultivated in Swiss albino mice with 50–60% parasitemia before collecting the blood. The P. berghei-infected blood, mainly at the trophozoite stage, was then passed through CF-11 cellulase columns to remove all white blood cells and platelets. Both parasites were freed of their host red cells by incubating in an equal volume of 0.15% saponin in RPMI 1640 medium at 37⬚C for 20 min. The intact parasites were then washed at least 4 times (8000g, 10 min) with ice-cold phosphate-buffered saline, pH 7.4, containing 1 mM phenylmethylsulfonyl fluoride (Krungkrai et al. 1990). Mitochondria were prepared from the intact parasites as previously described (Fry and Beesley 1991; Krungkrai 1995; Krungkrai et al. 1993). Briefly, the parasites were homogenized in a medium containing 75 mM sucrose, 225 mM mannitol, 5 mM MgCl2, 5 mM KH2PO4, 1 mM ethylene glycol-bis(␤-aminoethylether)-N,N,N ⬘,N ⬘-tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, and 5 mM N-2-hydroxyethylpiperazineN ⬘-2-ethanesulfonic acid (pH 7.4); the mitochondria in the homogenate

55 were purified by differential centrifugation and 22% Percoll density gradient centrifugation. The mitochondria were stored at ⫺196⬚C before enzyme NADH dehydrogenase purification. The purity of the mitochondria was monitored by examining their characteristics on transmission electron microscopy and measuring three marker enzymes, dihydroorotate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase (Krungkrai 1995; Krungkrai et al. 1993). Measurement of oxygen uptake by mitochondria. The rate of oxygen uptake by the mitochondrial organelles was measured polarographically by using a Clark-type oxygen electrode and a YSI oxygen monitor. Calibration of oxygen concentration during mitochondrial respiration was performed according to a procedure of Robinson and Cooper (1970). Before starting oxygen uptake measurement, the freshly prepared mitochondria in homogenizing medium were permeabilized by sonication on ice for six 10-s bursts in a pulse mode at 10% duty cycle using a Bendelin Sonopus HD2070 sonicator (Rickwood et al. 1987). The oxygen uptake reaction in a total volume of 3 ml was followed for 3–5 min at 37⬚C with a temperature-controlled circulator. Compounds affecting mammalian mitochondrial functions, e.g., NADH, adenosine 5⬘-diphosphate (ADP), rotenone (Hatefi 1985), and plumbagin (5hydroxy-2-methyl-1,4-naphthoquinone), were tested by being added to the oxygen uptake chamber, and the rate of oxygen consumption was then followed for the next 3–5 min. Respiratory rate by the mitochondria was expressed as nmol oxygen utilized/min/mg protein. ADP:O ratio of mitochondrial oxidative phosphorylation was calculated according to the method of Estrabrook (1967). Purification of NADH dehydrogenase on fast protein liquid chromatographic system. The mitochondria, stored at ⫺196⬚C and pooled from five to seven different preparations of either P. falciparum or P. berghei parasites, were treated with an equal volume of 0.2% n-octyl glucoside and then stirred at 4⬚C for 1 h. The supernant fluid, collected after centrifugation of the mitochondrial extract at 100,000g for 1 h, was concentrated and applied to a Pharmacia Mono Q anion-exchange fast protein liquid chromatographic column which had been equilibrated with 5 mM phosphate buffer, pH 8.0, containing 0.1% n-octyl glucoside, 1 mM phenylmethylsulfonyl fluoride, and 5 mM ethylenediaminetetraacetic acid (buffer A). The column was then washed with buffer A and proteins were eluted with a linear gradient from 0 to 600 mM KCl over 30 min at a flow rate of 1 ml/min. The 1.0-ml fractions were collected and then measured for both protein and enzyme activity. The active fractions from the Mono Q column were pooled, dialyzed against buffer A, concentrated, and then applied to a Pharmacia Superose 6 gel-filtration FPLC column equilibrated with buffer A containing 150 mM KCl. The enzyme was eluted with this buffer over 45 min at a flow rate of 0.5 ml/min, the 0.5-ml fractions were collected and assayed for the enzyme activity. The active fractions were pooled and stored as aliquots at ⫺196⬚C. The Superose 6 column was calibrated with Bio-Rad molecular mass markers: thyroglobulin (670 kDa), immunoglobulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), and vitamin B12 (1.35 kDa). Enzyme assay and kinetic studies. The activity of NADH dehydrogenase during purification was measured by following 2,6-dichlorophenolindophenol reduction at 600 nm and 37⬚C using a Shimadzu 1601 spectrophotometer equipped with a temperature-controlled system as essentially described (Galante and Hatefi 1978). The enzymatic reaction, in a total volume of 1.0 ml, contained 50 mM phosphate buffer, pH 8.0, 0.1% n-octyl glucoside, 0.1 mM NADH, 0.1 mM CoQ (2,3dimethoxy-5-methyl-1,4-benzoquinone), 0.145 mM of 2,6-dichlorophenolindophenol. The reaction was started by adding enzyme into the reaction and followed for at least 5 min. The enzyme volume used for

56 the reactions was varied from 10 to 50 ␮l, depending on the activity of enzyme in each preparation. The specific activity of the enzyme was calculated using an extinction coefficient of 21,000 M⫺1 cm⫺1 for 2,6-dichlorophenolindophenol at 600 nm. For kinetic studies, the NADH dehydrogenase activity of the partially purified enzyme was measured with NADH oxidation (0.1 mM) at 340 nm, using an extinction coefficient of 6200 M⫺1 cm⫺1 and using 10 ␮M CoQ8 or CoQ10 as terminal electron acceptor for parasite and mouse liver enzymes, respectively (Galante and Hatefi 1978). Kinetic constants, Km and Vmax, were determined by fitting data to the Michaelis–Menten equation using nonlinear regression of Enzfitter program (Elsevier Biosoft). I50 was defined as the concentration of compound having 50% inhibitory effect against the partially purified enzyme. Other methods. Mammalian mitochondria were prepared from Swiss albino mouse liver as described previously (Rickwood et al. 1987). Intact enzyme of complex I from both malarial parasites and mouse liver was prepared as whole mitochondrial extract by solubilizing the purified mitochondria with an equal volume of 0.2% n-octyl glucoside at 4⬚C for 1 h. Protein concentrations were determined by the method of Bradford (1976) and using bovine serum albumin as standard. SDS–PAGE was performed on a Bio-Rad minislab gel apparatus with a 5% acrylamide stacking gel and 10% acrylamide running gel in the discontinuous buffer system of Laemmli (1970). The gels were stained with Coomassie blue R.

RESULTS AND DISCUSSION

It has been shown that P. falciparum in the asexual blood stage is a microaerophillic organism and favors its growth and development in very low oxygen tension, e.g., 0.5–3% oxygen (Scheibel et al. 1979). The mitochondrial organelles from both P. falciparum and P. berghei have been shown to be morphologically heterogeneous in various developmental stages (Fry and Beesley 1991; Krungkrai 2000; Krungkrai et al. 1993, 1999b, 2000). The function of these organelles in the parasites remains unknown. They might have functions for energy production (Krungkrai 2000; Krungkrai et al. 2000, Uyemura et al. 2000) and electron sink of the de novo pyrimidine biosynthetic pathway through dihydroorotate dehydrogenase (Gutteridge et al. 1979; Krungkrai et al. 1991; Krungkrai 1995). Based on the lines of evidence for the oxygen uptake by free parasites of P. falciparum (Krungkrai et al. 1999a,b, 2000) and P. berghei (Uyemura et al. 2000), the mitochondrial ETS of the malarial parasites during the asexual stage has been proposed to contain respiratory complexes I, II, III, and IV. In this study, NADH dehydrogenase of complex I was partially purified from mitochondria of P. falciparum, P. berghei, and mouse liver. Some kinetic properties were also compared to those of the intact complex I enzyme. The mitochondrial organelles were isolated and checked for their

KRUNGKRAI ET AL.

purity based on both the marker enzyme assays (Krungkrai 1995; Krungkrai et al. 1993) and the morphology using transmission electron microscopic observation. The purified organelles contained the three marker enzymes (dihydroorotate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase) with an average 25-fold enrichment over the parasite homogenate. The tubular and noncristate structures were demonstrated in the isolated mitochondria from both parasites, suggesting typical properties of these organelles. The freshly prepared mitochondria were then used for oxygen consumption measurements. It was found that the mammalian complex I inhibitor rotenone, at a concentration of 0.33 mM, had strong inhibitory effect against the oxygen uptake by the mitochondria isolated from P. falciparum, P. berghei, and mouse liver, with 72, 74, and 80% inhibition, respectively (Table I). The rates of oxygen uptake by the mitochondria isolated from both parasites were found to be relatively low, compared with the mammalian mouse liver mitochondria. The parasite mitochondria did not respond to ADP stimulation when using NADH as substrate. The mouse liver organelles were susceptible to the stimulating effect of both NADH and ADP additions, the ADP:O ratio was calculated to be ⬃3. This result also indicated the intactness of mitochondria during oxygen consumption measurement, since the amount of oxygen utilized was proportional to the amount of ADP phosphorylated to ATP when the organelles were at respiratory state 3 for energy production (Rickwood et al. 1987). The inhibitor of complex IV, KCN, at a concentration of 1 mM, had completely inhibitory effect against oxygen uptake by all mitochondria preparations (Table I). These results

TABLE I Rate of Oxygen Consumption by Isolated Mitochondria from Plasmodium falciparum, Plasmodium berghei, and Mouse Liver Oxygen consumption a (nmol/min/mg protein) Condition

P. falciparum

P. berghei

Mouse liver

0.33 mM NADH 0.3 mM ADP b 0.33 mM Rotenone b 1.0 mM KCN b

1.8 ⫾ 0.1 1.6 ⫾ 0.2 0.5 ⫾ 0.1 0

2.3 ⫾ 0.2 2.4 ⫾ 0.2 0.6 ⫾ 0.1 0

26.5 ⫾ 4.2 74.8 ⫾ 6.5 5.4 ⫾ 0.4 0

Values are mean ⫾ SD, obtained from five to six separate experiments using mitochondria prepared from malarial parasites and mouse liver with approximately 70–200 ␮g protein and ⬃30–40 ␮g per 3 ml oxygen uptake reaction, respectively. The medium used for the reaction assay was described under Materials and Methods. b ADP or rotenone or KCN at final concentrations as indicated was added 3 min after the addition of NADH into the reaction. a

NADH DEHYDROGENASE OF MALARIAL PARASITES

show a clear difference between the parasite and the mammalian mitochondria, the latter having a tight coupling oxidative phosphorylation for energy production. It is suggested that the mitochondria of the parasites are in the underdeveloped forms, but they are biochemically active at least with regard to oxygen consumption to maintain membrane potential and to electron sinks of the pyrimidine biosynthesis. This phenomenon may be related to the evidence that they do not have a functional citric acid cycle (Sherman 1979; Scheibel 1988), and they are acristate organelles (Langreth et al. 1978). This observation is consistent with the recent data on the existence of the respiratory complex I and limited metabolic activities in P. falciparum trophozoites (Krungkrai et al. 1999a,b) and P. berghei trophozoites (Uyemura et al. 2000). In addition, our results demonstrate the presence of catalytic subunits NADH dehydrogenase of complex I in the mitochondrial ETS from both malarial parasites. We have further characterized the NADH dehydrogenase by subjecting the detergent extract of the mitochondrial organelles isolated from P. falciparum, P. berghei, and mouse liver to the anion-exchange Mono Q FPLC columns (Fig. 1). The elution profiles in Fig. 1 were plotted from different runs of mitochondrial extract which had been separately prepared from these three organisms. Major activities (70%) of the enzymes from the parasites were eluted at low salt (⬃80–100 mM KCl, fractions 3–7), whereas the mammalian enzyme was eluted at high salt as a sharp peak (⬃420 mM KCl, fractions 21–22). The minor activities (30%) were also detected only in the parasites at ⬃150 and 300 mM KCl (Fig. 1). These minor ones were not further studied. The

57 pooled fractions of the major activities passed through the Mono Q columns were then purified through the gel filtration Superose 6 FPLC column (Fig. 2). The result in Fig. 2 is a representative eluting profile of P. falciparum enzyme. It was found that the enzymes from the parasites and the mouse liver mitochondria were eluted at positions of ⬃130 (both P. falciparum and P. berghei), and ⬃240 kDa (data not shown), respectively. When subjecting the partially purified enzymes to SDS–PAGE analysis, 2 major protein bands with molecular masses of 38 and 33 kDa were obtained from the parasites (Fig. 3, lanes a and b), whereas the mouse liver had ⬃12 protein bands (data not shown). The results of purifications of the enzymes from P. falciparum, P. berghei, and mouse liver mitochondria are summarized in Table II. Purification of these enzymes from different sources resulted in relatively low yields of ⬃2–4% and ⬃6 to 15fold. All three enzymes were found to be extremely labile. The enzyme activity decreased by more than 80% when stored overnight at 0⬚C, but was more stable when stored at ⫺196⬚C than at ⫺20⬚C. Since the purification of these enzymes to homogeneity was hampered by their labile properties, many attempts have been made to reactivate the enzyme activity. It was found that the addition of 10 ␮M flavin mononucleotide, as a coenzyme, had an effect on reactivating the mouse liver enzyme activity, but it showed no effect on the parasite enzyme. The difference in the stimulating effect by this coenzyme is still unknown. However, these results show the differences in physical properties in the parasite and mammalian enzymes, e.g., molecular mass, multimeric

FIG. 1. FPLC chromatogram of Plasmodium falciparum, Plasmodium berghei, and mouse liver NADH dehydrogenase activities purified through the Mono Q anion-exchange column eluting with KCl gradient. The eluting profile of each enzyme from three organisms was plotted on the same graph. The symbols and the amount of protein used were as follows: 䡵, P. falciparum (9.4 mg protein); 䉱, P. berghei (14.1 mg protein); 䢇, mouse liver (9.7 mg protein). The enzyme was collected as 1.0-ml fractions. The broken line indicates the linear gradient of KCl.

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FIG. 2. FPLC chromatogram of Plasmodium falciparum NADH dehydrogenase purified through the Superose 6 gel-filtration column. The symbols used were as follows: 䢇, enzyme activity; 䡬, mg protein per 0.5-ml fraction. The arrows indicate eluting positions of protein markers with Mr indicated on the top.

formation, number of subunits, and eluting behavior on the anion-exchange chromatographic column. The malarial complex I may contain limited subunits for catalysis, e.g., NADH dehydrogenase, that function only for transhydrogenation from NADH to CoQ in the mitochondrial membrane. This phenomenon is similar to the recent

FIG. 3. SDS–PAGE of Plasmodium falciparum and Plasmodium berghei NADH dehydrogenase purified through the Superose 6 gelfiltration column. The gel was stained with Coomassie blue R. Lane a, 5 ␮g of the P. falciparum enzyme; lane b, 5 ␮g of the P. berghei enzyme; lane c, control SDS-sample buffer; lane d, known proteins with Mr indicated on the right.

findings that succinate dehydrogenase of complex II in P. falciparum has only two major catalytic subunits, flavoprotein and iron–sulfur protein (Suraveratum et al. 2000; Takeo et al. 2000). Since P. falciparum and P. berghei have no complex I genes encoded by mitochondrial DNA (Feagin and Drew 1995), by using the amino acid sequences of Ascaris suum, Caenorhabditis elegans, Escherichia coli, and Saccharomyces cerivisae NADH dehydrogenase and searching the Sanger Center for malaria database, it is now possible to obtain putative NADH dehydrogenase gene of complex I (chromosome 5) and to deduce protein-coding sequence with 286 amino acids (data not shown). This enzyme flavoprotein homolog, a calculated Mr of 33.2 kDa from the deduced amino acid sequence, may be the 33-kDa protein of the two major bands on the SDS–PAGE (Fig. 3). Gene encoding the 38-kDa subunit was not identified. Cloning and expression of the NADH dehydrogenase in P. falciparum need to be further investigated. Kinetic analyses were determined for comparison between the partially purified enzymes after the Superose 12 FPLC column and the intact enzymes (complex I) were prepared in 0.2% n-octyl glucoside-treated mitochondria extract from both malarial parasites and mouse liver. It was found that the partially purified malarial enzymes required both NADH and CoQ for their maximal activity, and they followed Michaelis–Menten kinetics for each substrate. CoQ8 and CoQ10 were used as naturally occurring coenzymes for the enzymes isolated from the parasites (Wan et al. 1974) and mouse liver (Hatefi 1985), respectively. The Km values for both substrates of the partially purified NADH dehydrogenases

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TABLE II Purification of NADH Dehydrogenases from the Isolated Mitochondria of Plasmodium falciparum (Column I), Plasmodium berghei (Column II), and Mouse Liver (Column III) a Total activity (mU) b

Protein (mg) Step 1. Mitochondria 2. Mono Q 3. Superose 6

Specific activity (mU/mg protein)

Yield (%)

Fold purification

I

II

III

I

II

III

I

II

III

I

II

III

I

II

III

9.4 0.84 0.05

14.1 0.26 0.02

9.7 0.40 0.03

24.4 2.9 1.0

35.4 4.8 0.73

152.3 15.8 3.1

2.6 6.0 20.0

2.5 18.5 36.5

15.7 39.5 103.3

100 11.9 4.1

100 13.6 2.1

100 10.4 2.0

1 2.3 7.7

1 7.4 14.6

1 2.5 6.6

a The mitochondria were isolated as follows: 3.0 ml packed host red cell-free P. falciparum (255 mg protein), 3.5 ml packed host red cell-free P. berghei (306 mg protein), and 3 g wet weight mouse liver (protein was not determined). b The mU of enzyme activity is expressed as nmol/min at 37⬚C.

from the three sources were determined from the plots of the Enzfitter software program and the results are shown in Table III. The Km values for NADH of the purified NADH dehydrogenases were found to be identical to those of the intact complex I enzyme among the three sources. In addition, the Km values for CoQ8 of both parasites were similar in the purified and intact complex I enzymes. In contrast, the NADH dehydrogenase from mouse liver had ⬃5 times higher Km values for CoQ10 than the complex I (Km⫽ 0.05 ␮M). The Vmax values of the malarial NADH dehydrogenases were approximately 7–10 times lower than that of the mammalian enzyme. The complex I inhibitor rotenone had inhibitory effect on P. falciparum, P. berghei, and mouse liver NADH dehydrogenase enzymes (Table III) and the oxygen uptake by isolated mitochondria (Table I). It was found that the I50 value of rotenone for the intact complex I from mouse liver (1.2 ⫾ 0.3 ␮M, n ⫽ 3) was 7 times lower than that of the purified enzyme, whereas the I50 values were not different in the enzymes prepared as intact complex I and purified forms of the malarial parasites. Compared to the beef heart muscle mitochondria, the intact complex I is 90% inhibited by rotenone at 2.5 ␮M (Ragan et al. 1987), but

the purified NADH dehydrogenase showed much less effect to rotenone inhibition (Galante and Hatefi 1978). This is consistent with our observation and with the recently characterized complex I, rotenone-sensitive NADH:ubiquionone oxidoreductase, in Trypanosoma brucei (Fang et al. 2001). Furthermore, the parasite enzyme was more sensitive to plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; a CoQ analog) inhibition than the mammalian enzyme (Table III). Plumbagin was found to completely inhibit the oxygen uptake by parasite mitochondria at a concentration of 5 ␮M (Suraveratum et al. 2000). Our results show some differences in kinetic properties and sensitivity to the inhibitor in the parasite and the mammalian enzymes, e.g., Km , Vmax, and I50 of plumbagin. This is consistent with previous observation that both rotenone (Ginsburg et al. 1986; Krungkrai et al. 1999a) and plumbagin (Suraveratum et al. 2000) exhibit antimalarial activity against in vitro growth of P. falciparum with 50% inhibitory concentrations of 17 and 0.3 ␮M, respectively. This suggests that rotenone and plumbagin, the inhibitors of the mitochondrial respiratory complex I, are targeted on the malarial NADH dehydrogenase. Nevertheless, plumbagin may have additional targeting enzymes in

TABLE III Enzyme Kinetic and Inhibitory Parameters of Plasmodium falciparum, Plasmodium berghei, and Mouse Liver Partially Purified Mitochondrial NADH Dehydrogenases Source

K mNADH (␮M)

K mCoQa (␮M)

Vmax (nmol/min/mg)

I rotenone (␮M) 50

P. falciparum P. berghei Mouse liver

5.2 ⫾ 0.6 2.7 ⫾ 0.4 1.3 ⫾ 0.4

0.82 ⫾ 0.06 0.55 ⫾ 0.08 0.24 ⫾ 0.02

54.4 ⫾ 6.5 72.6 ⫾ 8.4 494.4 ⫾ 34.7

12.5 ⫾ 3.2 10.6 ⫾ 2.2 8.4 ⫾ 1.4

a

b

plumbagin

I 50

(␮M)

6.5 ⫾ 1.3 5.3 ⫾ 0.8 35.8 ⫾ 4.2

CoQs used were as follows: CoQ8 for the parasite enzymes; CoQ10 for the mouse liver enzyme. Values are mean ⫾ SD, taken from five to six separate experiments of the enzyme preparations using the amount of protein in the range of 0.1–1.0 ␮g. b

60 P. falciparum, e.g., succinate dehydrogenase of complex II in which Km value of CoQ is four-fold less than that of the NADH dehydrogenase reported in this study (Suraveratum et al. 2000). It is noted here that the compound having a different structure from rotenone and plumbagin, e.g., dimethylbiguanide, is also targeted on the mitochondrial ETS complex I of a mammalian system (El-Mir et al. 2000). Two antimalarial drugs, artemisinin and atovaquone, having 50% inhibitory concentrations of ⬃1–5 nM levels on P. falciparum in in vitro growth (Krungkrai 2000, Krungkrai et al. 1999b), showed moderate inhibitory effect against the parasite enzyme with I50 of 60 and 22.5 ␮M, respectively. Previously we reported that artemisinin and atovaquone inhibited the mitochondrial oxygen consumption in P. falciparum at concentrations of 5 ␮M and 50 nM, respectively (Krungkrai et al. 1999a). The de novo pyrimidine biosynthetic pathway and mitochondrial ETS in the asexual stage of malarial parasites is associated (Gutteridge et al. 1979; Krungkrai et al. 1991; Krungkrai 1995). The linked pathways are considered to be possible targets for some existing drugs, e.g., tetracycline (Prapunwattana et al. 1988), atovaquone (Krungkrai 1995; Krungkrai et al. 1997; Srivastava et al. 1997), and artemisinin (Kawai et al. 1993; Krungkrai et al. 1999a). Based on our findings and other evidence, it is proposed that P. falciparum and P. berghei may have functional mitochondria containing four enzyme activities of NADH dehydrogenase of complex I, succinate dehydrogenase of complex II, ubiquinol-cytochrome c reductase of complex III, and cytochrome c oxidase of complex IV. The mitochondrial ETS may contribute significantly to synthesis of pyrimidine nucleotides and to energy metabolism of the parasite. In conclusion, the P. falciparum and P. berghei NADH dehydrogenase is indeed a unique enzyme: (1) it is physically and kinetically different from the enzyme of the mammalian host, (2) the potent antimalarial plumbagin has strong inhibitory effect on the enzyme, and (3) it may be a potential target for selective chemotherapeutic agents.

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REFERENCES

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ACKNOWLEDGMENTS

We thank Nuchanat Wuttipraditkul and Sanya Kudan for their excellence technical assistance with parasite cultivation, animal handling, and enzyme purification. This work was partly supported by the UNDP/ World Bank/WHO Special Programme for Research and Training in Tropical Diseases and by the Thailand Research Fund.

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