- Email: [email protected]
Purification and characterization of Plasmodium falciparum succinate dehydrogenase
Molecular and Biochemical Parasitology 105 (2000) 215 – 222 www.elsevier.com/locate/parasitology
Purification and characterization of Plasmodium falciparum succinate dehydrogenase Nongluk Suraveratum a,1, Sudaratana R. Krungkrai b, Preecha Leangaramgul a, Phisit Prapunwattana a, Jerapan Krungkrai a,* a
Department of Biochemistry, Faculty of Medicine, Chulalongkorn Uni6ersity, Rama IV Road, Bangkok 10330, Thailand b Department of Biochemistry, Faculty of Science, Rangsit Uni6ersity, Patumthani 12000, Thailand Received 2 August 1999; received in revised form 28 September 1999; accepted 28 September 1999
Abstract 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 909 8 kDa and contained two major subunits with molecular masses of 55 9 6 and 3594 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 mM, and kcat 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 Ki values of 81, 13 and 12 mM, 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 mM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 mM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 mM. 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 mM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Plasmodium falciparum; Succinate dehydrogenase; Mitochondrial electron transport complex; Chemotherapeutic target; Antimalarial agent
Abbre6iations: CoQ, 2,3-dimethoxy-5-methyl-1,4-benzoquinone; ETS, electron transport system; Fp, flavoprotein; Ip, iron – sulfur protein; PMSF, phenylmethylsulfonyl fluoride; SDH, succinate dehydrogenase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TTFA, 2-thenoyltrifluoroacetone. * Corresponding author. Tel.: +66-2-252-4986; fax: + 66-2-256-4482. E-mail address: [email protected] (J. Krungkrai) 1 Present address: Unit of Biochemistry, Rajavithi Hospital, Rajavithi Road, Bangkok 10400, Thailand. 0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 9 ) 0 0 1 8 0 - 2
216
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
1. Introduction Succinate dehydrogenase (complex II, succinate: ubiquinone oxidoreductase, EC 1.3.99.1) is a tricarboxylic acid (Krebs) cycle enzyme that functions as a primary dehydrogenase, donating electrons to the aerobic and energy-generating respiratory chain in eukaryotic mitochondria and numerous prokaryotes [1]. The enzyme has been isolated and characterized from a number of prokaryotic [2–4] and eukaryotic sources [5,6]. Relatively little is known about the enzyme in the genus Plasmodium. During the asexual blood stage of the malarial parasites, they grow and mature within the erythrocytes of their hosts. The absence of cristate structure in mitochondrial organelle at this stage of the parasite has been demonstrated [7 – 10]. Energy requirement during this stage is provided by metabolizing glucose primarily by anaerobic glycolysis [11,12]. Only two Krebs cycle enzymes, malate dehydrogenase and isocitrate dehydrogenase, have been demonstrated in Plasmodium falciparum and Plasmodium knowlesi [13 – 15]. It has been shown that P. falciparum lack at least some of Krebs cycle enzymes, e.g. a-ketoglutarate dehydrogenase [16]. It has been clearly evident that the malaria parasites have mitochondrial electron transport system (ETS) and oxygen-requiring system that is necessary for growth and survival [6,8,11,12,17,18]. Recently the ETS complex III (ubiquinol-cytochrome c reductase) and complex IV (cytochrome c oxidase) have been purified and characterized in Plasmodium berghei and P. falciparum [9,19]. In this study, the succinate dehydrogenase (SDH) enzyme has been purified from the asexual blood stage, mainly trophozoite, of P. falciparum grown in vitro. Physical and kinetic properties of the purified enzyme are investigated. These properties observed in the malarial SDH are different from those in the mammalian mitochondrial enzyme.
2. Materials and methods
2.1. Culti6ation of malaria parasite
P. falciparum (a pyrimethamine resistant T9 line) was cultivated by a modification of the candle jar method of Trager and Jensen [20], using a 5% hematocrit of human erythrocytes type O suspended in RPMI 1640 medium supplemented with 25 mM Hepes, 32 mM NaHCO3 and 10% fresh human serum type O. The cultures started at low parasitemia ( 1–2%) were changed with the medium twice daily until the cultures had 30% parasitemia and then harvested for enzyme preparation. Synchrony of the culture was maintained by sorbitol procedure of Lambros and Vanderberg [21].
2.2. Purification of enzyme on fast protein liquid chromatographic system The parasites freed from the infected erythrocytes containing mostly trophozoite stage were washed and lysed according to the established procedure [22].The parasite homogenate containing 0.2% octyl glucoside was dialyzed, concentrated and loaded onto a Pharmacia Mono Q FPLC column equilibrated with 5 mM phosphate buffer, pH 8.0, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM EDTA. The column was then washed with the equilibrating buffer and proteins were eluted with a linear gradient from 0 to 600 mM KCl over 40 min at a flow rate of 1 ml min − 1. The 1.0-ml fractions were collected and then measured for SDH enzyme activity. The active fractions from the Mono Q column eluted at 70 mM KCl were dialyzed, concentrated and then loaded onto a Mono S cation-exchange FPLC column which was equilibrated with 5 mM phosphate buffer, pH 6.0, containing 1 mM PMSF and 5 mM EDTA. The column was washed with the same buffer and then eluted with linear gradient of the phosphate buffer from pH 6.0 to pH 8.0 at a flow rate of 1 ml min − 1. The eluates were collected into 38 fractions, each 1.0 ml fraction was determined for SDH activity and protein concentration. The eluates containing the enzyme activity from the Mono S column (at pH 6.3) were pooled,
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
concentrated and then applied to a Superose 6 gel filtration FPLC column equilibrated with 5 mM phosphate buffer, pH 8.0, containing 1 mM PMSF, 5 mM EDTA and 150 mM KCl. The enzyme was eluted with this buffer over 45 min at a flow rate of 0.5 ml min − 1, the 0.5-ml fractions were collected and assayed for the SDH activity. The active fractions were pooled and stored as aliquots at − 196°C. The Superose 6 column was calibrated with the following Pharmacia molecular mass markers: thyroglobulin (670 kDa), immunoglobulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa).
2.3. Enzyme assay and kinetic studies SDH activity was measured by following reduction of cytochrome c at 550 nm and 37°C using a Shimadzu 1601 spectrophotometer equipped with a temperature-controlled unit. The enzymatic reaction, in a total volume of 1.0 ml, contained 50 mM phosphate buffer, pH 8.0, 1 mM succinate, 0.01 mM 2,3-dimethoxy-5-methyl-1,4-benzoquinone (CoQ) and 0.1 mM cytochrome c. A specific inhibitor of SDH, 10 mM malonate, was also included in the enzymatic reaction for correcting non-specific dehydrogenase(s) activities in the parasite homogenate before purification steps. For kinetic studies, the SDH activity on the purified enzyme was measured with dichlorophenolindophenol as a terminal electron acceptor, the absorbance change at 600 nm was then monitored. Kinetic constants, Km and kcat, were determined by fitting data to the Michaelis – Menten equation using non-linear regression of Enzfitter program (Elsevier Biosoft). Inhibitor constants (Ki) were determined from Dixon’s plots. I50 was defined as the concentration of compound having 50% inhibitory effect against the purified enzyme.
2.4. Measurement of oxygen uptake by host cell-free P. falciparum The rate of oxygen uptake for a consistent number of host cell-free P. falciparum parasites was measured in a modified medium containing 75 mM sucrose, 225 mM mannitol, 5 mM MgCl2,
217
5 mM KH2PO4, 1 mM EGTA, 5 mM Hepes, pH 7.4 [9] by using a Clark-type oxygen electrode and YSI oxygen monitor according to the method of Robinson and Cooper [23]. The oxygen uptake reaction in a total volume of 3 ml was followed and recorded at 37°C with a temperature-controlled circulator.
2.5. In 6itro antimalarial test Antimalarial activity against P. falciparum in vitro was quantified by measuring % parasitemia in a 4-day culture in the presence of the tested compounds at various concentrations [24]. All compounds were tested in triplicate at each concentration used. The 50% inhibitory concentration (IC50) was defined as the concentration of the compound causing 50% inhibition of the parasite growth in a 4-day culture, compared with the compound-free control of the parasite culture.
2.6. Other methods Mammalian mitochondria were prepared from mouse liver as described previously [25]. Protein concentrations were determined by the method of Bradford [26] and using bovine serum albumin as standard. SDS-PAGE was performed on a BioRad minislab gel apparatus with a 5% acrylamide stacking gel and 10% acrylamide running gel in the discontinuous buffer system of Laemmli [27]. Nondenaturing-PAGE was performed by using the modified Laemmli’s method in the absence of SDS and 2-mercaptoethanol in all reagents used, and 10% acrylamide running gel was applied. The gels were stained with Coomassie Blue R and visualized by a Bio-Rad molecular analyst PC software image analysis.
3. Results Using the cytochrome c reduction assay, the SDH activity in the parasite homogenate was approximately 309 4% of total dehydrogenase activity. It was noted that after FPLC steps the SDH activity was completely inhibited in the pres-
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
218
Table 1 Purification of succinate dehydrogenase (SDH) from 3.0-ml of packed host erythrocyte-free Plasmodium falciparum Step
Total protein (mg) Total activity (mU)a
Specific activity (mU mg−1)
Yield (%)
Fold purification
1. 2. 3. 4.
139.56 15.04 3.96 0.08
1.37 5.38 6.17 33.13
100 42.3 12.8 1.4
1 3.9 4.5 24.2
Homogenate Mono Q FPLC Mono S FPLC Superose 6 FPLC a
191.08 80.88 24.42 2.65
The mU of enzyme activity is expressed as nmol min−1 at 37°C.
ence of 10 mM malonate. The results of a typical purification of the malarial SDH enzyme are summarized in Table 1. Purification of the parasite enzyme resulted in relatively low yield of 1% and a 24-fold purification. The parasite enzyme was 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 that of − 20°C. Many attempts have been made to reactivate the parasite enzyme activity, e.g. addition of stabilizers (0.1 mM 2-mercaptoethanol or 10 mM 1,4-dithiothreitol or 1 mM succinate) or flavin coenzymes (0.01 mM of FADH2 or FMN). However, these additives had no effect on the reactivating and also stabilizing the enzyme activity during purification. The purification of the malarial enzyme was hampered by its lability property, however, eight purifications were performed. Using nondenaturing PAGE the purified enzyme (eluted from the Superose 6 gel filtration FPLC column, Table 1) exhibited one major band (Fig. 1A). The purified enzyme was then subjected to SDS-PAGE to determine number and molecular mass of enzyme subunits (Fig. 1B). There were few faint bands observed at positions of 90 and 13 –20 kDa when 20 mg of the purified enzyme was loaded onto the gel (Fig. 1B, lane a). The results obtained in Fig. 1 suggest that the parasite enzyme had two major subunits with apparent molecular masses of 5596 and 359 4 kDa (n = 8). The enzyme had a native molecular mass of 909 8 kDa (n= 8) based on the eluting position on the Superose 6 gel filtration FPLC column (Fig. 2).
Kinetic analyses of the parasite SDH demonstrated that the enzyme required both succinate and CoQ for its maximal activity, and followed Michaelis–Menten kinetics for each substrate. The Km and kcat values for both substrates were determined from the plots of the Enzfitter software program and the results are shown in Table 2. The catalytic efficiency of the malarial enzyme, calculated as kcat/Km, was found to be relatively low (Table 2). By using Dixon’s plot, malonate and oxaloacetate, two analogs of the substrate succinate, were found to be effective competitive
Fig. 1. (A) Nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis of purified Plasmodium falciparum succinate dehydrogenase eluted from the Superose 6 gel filtration FPLC column. The amount of protein loaded onto the gel of lanes a and b was 10 and 20 mg, respectively. The arrow indicates position of a major protein band on the gel. (B) Sodium dodecyl sulfate (SDS)-PAGE analysis of purified P. falciparum succinate dehydrogenase. The amount of protein loaded onto the gel of lanes a and b was 20 and 10 mg, respectively. The bars indicate positions of molecular mass marker proteins: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa).
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
219
Fig. 2. FPLC chromatogram of Plasmodium falciparum succinate dehydrogenase on the Superose 6 gel filtration column. The arrows show eluting positions of calibrated molecular mass proteins. The symbols used were as follows: , total enzyme activity per 0.5-ml fraction; , amount of protein per 0.5-ml fraction. Table 2 Enzyme kinetic and inhibitory constant parameters of Plasmodium falciparum succinate dehydrogenase (SDH) Compound
Km (mM)
kcat (min−1)
Ki (mM)
kcat/Km (M−1 s−1)
Substrates: Succinate CoQ
3.019 0.05a 0.209 0.03
0.119 0.01 0.069 0.01
– –
600 5000
Inhibitors: Malonate Oxaloacetate Fumarate a
13.02 9 0.49 12.06 9 0.81 80.99 9 3.07
Values are mean 9 S.D., taken from five to six separate experiments of the enzyme preparations.
inhibitors of the parasite enzyme. Fumarate, the enzymatic product, showed an inhibitory effect against the malarial SDH (Table 2). The known mammalian ETS complex II inhibitor 2-thenoyltrifluoroacetone (TTFA) had no inhibitory effect against both the parasite enzyme activity and the oxygen uptake by the host cellfree parasites at concentration of 50 mM. The oxygen uptake by these parasites in the assay system as described in Section 2 were found to be 1.56 90.23 nmol min − 1 mg − 1 (n = 3). The parasite’s oxygen uptake showed relatively resistance to TTFA inhibition, only 10% inhibition was observed at 100 mM, whereas TTFA at this con-
centration was found to inhibit more than 90% of the mouse liver mitochondrial oxygen uptake. Chloroquine (100 mM), artemisinin (5 mM) and atovaquone (5 mM) showed no inhibitory effect against the parasite enzyme activity. Interestingly, the parasite SDH activity was sensitive to inhibition of 5-hydroxy-2-methyl-1,4-naphthoquinone (or plumbagin), a CoQ analog, with an I50 of 5 mM. Furthermore, plumbagin was found to completely inhibit the oxygen uptake by the parasite at a concentration at 0.88 mM (or 165 ng ml − 1). It was also demonstrated that it had antimalarial activity against in vitro growth of P. falciparum with an IC50 of 0.27 mM (or 50.8 ng ml − 1).
220
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
4. Discussion The present findings clearly demonstrate that the malaria parasite P. falciparum possesses significant SDH activity. This is the first report on the purification and characterization of the important enzyme complex II responsible for mitochondrial ETS and Krebs cycle in the parasite. Our attempts on the biochemical purifications were hampered by the markedly labile and low specific activity of the malarial enzyme. It is generally accepted that the active site of the enzyme in various sources contains highly reactive sulfhydryl group that participates in the first step of succinate oxidation [28], the sulfhydryl group may be directly involved in the inactivation resulting in an unstable enzyme. This led one to use high amounts of parasites as the starting material to overcome the extremely low yield and purification. The malarial SDH has been purified to near homogeneity (Fig. 1). It had an apparent molecular mass of 90 kDa (Fig. 2) and contained 2 subunits with molecular masses of 55 kDa (Fp, flavoprotein subunit) and 35 kDa (Ip, iron – sulfur protein subunit). Similar results are also obtained from a rodent parasite P. berghei with Fp subunit 63 kDa and Ip subunit 35 kDa [Krungkrai et al. unpublished results]. However, faint bands at molecular masses of 13 – 20 kDa observed on the SDS-PAGE analysis of the enzyme preparation may be two subunits of low molecular masses of cytochrome b which are 15 – 20 and 13 – 16 kDa and reported in both bacterial and beef heart mitochondrial enzymes respectively [2,29,30]. The cytochrome b subunits of the ETS complex II appear to be essential for the assembly of the Fp and Ip subunits of SDH with the inner membrane of mitochondria [30]. The putative membrane anchoring and electron transferring functions of the two smaller subunits of cytochrome b in the malarial SDH enzyme needs to be further characterized. The molecular mass and the Fp and Ip subunits structure of the malarial enzyme were quite different to the SDH obtained from both Escherichia coli and mitochondrial beef heart. In E. coli the enzyme had a molecular mass of 118 kDa based on the nucleotide sequencing
data calculation, which consisted of Fp subunit 64 kDa, Ip subunit 28 kDa and two smaller subunits 19.5 and 17.5 kDa based on the SDSPAGE analysis [2]. The mitochondrial enzyme of beef heart had a molecular mass of 140 kDa and consisted of Fp subunit 70 kDa, Ip subunit 27 kDa and two cytochrome b subunits 15.5 and 13.5 kDa [5,6,29–31]. The catalytic efficiency of the malarial SDH (kcat/Km 600–5000 M − 1 s − 1) was not much different to another two enzymatic complexes of the parasite mitochondrial ETS, complex III ubiquinol-cytochrome c reductase (kcat/Km 12 000 M − 1 s − 1) and complex IV cytochrome c oxidase (kcat/Km 1300 M − 1 s − 1), which have been recently reported [19]. It is noted that the catalytic efficiency of the parasite enzyme is much lower than those of the bacterial and mammalian enzymes [2,5,6]. Several of the kinetic properties of the malarial enzyme differed from those reported for SDH from several other sources. The Km for succinate of the P. falciparum enzyme is 3 mM which compares with 20 mM measured for the E. coli SDH [2], 41 mM for the P. berghei SDH [Krungkrai et al. unpublished results], and 71 mM for the beef heart mitochondrial enzyme [6]. In addition, the Km for CoQ of the P. falciparum SDH is 0.2 mM which compares with 4.2 mM measured for the E. coli enzyme [2]. The malarial enzyme is also sensitive to the competitive inhibitors which have Ki values of 12 mM for oxaloacetate, 13 mM for malonate and 81 mM for fumarate, the enzymatic product. The Ki values for malonate of the beef heart mitochondrial and E. coli enzymes are 0.25 [6], and 200 mM [2], respectively. The Ki values for oxaloacetate and fumarate of the beef heart mitochondrial enzyme are reported to be 1.5 mM and 1.3 mM, respectively [31]. These Km and Ki values suggest that the active site of the malarial SDH is different from the reported enzymes in various organisms. This difference in active site was supported by a comparison of the classical mammalian mitochondrial enzyme inhibitor TTFA [1] with the enzymes from P. falciparum, E. coli [2], and beef heart mitochondria [6]. In contrast to the mammalian mitochondrial enzyme, the bacterial and malarial enzymes are relatively insensitive to
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
TTFA inhibition. Furthermore, when the oxygen uptake was measured in the malarial parasite and the mouse liver mitochondria, the parasite’s oxygen uptake was relatively resistant to the TTFA inhibition, as compared to the sensitivity to TTFA inhibition of the liver mitochondria. However, TTFA is reported to have relatively low inhibitory effect against the growth of P. falciparum in in vitro culture [17]. Interestingly, the CoQ analog plumbagin may exert its potent antimalarial effect through inhibition of the parasite SDH enzyme and the oxygen uptake activities. It should be also noted here that the parasite enzyme is not the target for the currently used antimalarial drugs, e.g. chloroquine, artemisinin and atovaquone. More recently, atovaquone is reported to specifically inhibit the parasite ETS complex III ubiquinol-cytochrome c reductase with an I50 of 5 nM [19]. During the progress of this work, Takeo and Kita have submitted the nucleotide sequences encoding both Fp and Ip subunits of P. falciparum SDH at GenBank (accession numbers: D86573 and D86574). The deduced amino acid sequences of both Fp and Ip subunits seem to have also unique properties. The calculated molecular masses of the genes of Fp subunit 69 kDa and Ip subunit 38 kDa were rather bigger than the proteins characterized in this report. It is possible that the parasite mitochondrial proteins synthesized in cytosol containing organelle targeting signal may be cleaved during translocation into mitochondrion, but verification of this will be necessary. Nevertheless, difference in the molecular mass has also been observed in the mitochondrial dihydroorotate dehydrogenase of P. falciparum between the deduced amino acid sequence from the nuclear gene [32] and the purified enzyme [8]. The P. falciparum SDH enzyme is indeed a unique enzyme. First, the parasite enzyme is physically and kinetically different from the enzymes of the mammalian tissues and the bacterial system. Second, the classical mammalian mitochondrial enzyme inhibitor 2-thenoyltrifluoroacetone shows no inhibitory effect on the parasite enzyme. Third, the potent antimalarial plumbagin has strong inhibitory effect on the enzyme. These
221
unique properties suggest that the SDH enzyme of the malarial parasites may be a potential target for selective chemotherapeutic agents.
Acknowledgements We thank Rachanok Kanjanarithisak and Sanya Kudan for their technical assistance with parasite cultivation, animal handling and enzyme purification. A part of this work was the M.S. thesis of N. Suraveratum. This work was supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. J. Krungkrai is grateful for a career development award from the National Science and Technology Development Agency of Thailand.
References [1] Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 1985;54:1015 – 69. [2] Kita K, Vibat CRT, Meinhardt S, Guest JR, Gennis RB. One-step purification from Escherichia coli of complex II (succinate:ubiquinone oxidoreductase) associated with succinate reducible cytochrome b556. J Biol Chem 1989;264:2672– 7. [3] Moll R, Schafer G. Purification and characterization of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius. Eur J Biochem 1991;207:593 – 600. [4] Reddy TLP, Weber MM. Solubilization, purification and characterization from membranes of M. phlei. J Bacteriol 1986;167:1 – 6. [5] Hatefi Y, Stiggall DL. Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol 1978;53:21 – 7. [6] Tushurashvili PR, Gavrikova EV, Ledenev AN, Vinogradov AD. Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. Biochim Biophys Acta 1985;809:145 – 59. [7] Fry M, Beesley JE. Mitochondria of mammalian Plasmodium spp. Parasitology 1991;102:17 – 26. [8] Krungkrai J. Purification, characterization and localization of mitochondrial dihydroorotate dehydrogenase in Plasmodium falciparum, human malaria parasite. Biochim Biophys Acta 1995;1243:351– 60. [9] Krungkrai J, Krungkrai SR, Bhumiratana A. Plasmodium berghei: partial purification and characterization of the
222
[10]
[11]
[12] [13]
[14] [15]
[16]
[17]
[18]
[19]
[20] [21]
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222 mitochondrial cytochrome c oxidase. Exp Parasitol 1993;77:136 – 46. Langreth SG, Jensen JB, Reese RT, Trager W. Fine structure of human malaria in vitro. J Protozool 1978;25:443 – 52. Scheibel LW. Plasmodial metabolism: carbohydrate. In: Wernsdorfer WH, McGregor I, editors. Malaria, vol. I. New York: Churchill Livingstone, 1988:171–217. Sherman IW. Biochemistry of Plasmodium (malaria parasites). Microbiol Rev 1979;43:453–95. Lang-Unnash N. Purification and properties of Plasmodium falciparum malate dehydrogenase. Mol Biochem Parasitol 1992;50:17 – 26. Lang-Unnash N. Plasmodium falciparum: antiserum to malate dehydrogenase. Exp Parasitol 1993;80:357–9. Sahni SK, Saxena N, Puri SK, Dutta GP, Pandey VC. NADP-specific isocitrate dehydrogenase from the simian malaria parasite Plasmodium knowlesi: partial purification and characterization. J Protozool 1992;39:338–42. Blum JJ, Ginsburg H. Absence of alpha-ketoglutarate dehydrogennase activity and presence of CO2-fixing activity in Plasmodium falciparum grown in vitro in human erythrocytes. J Protozool 1984;34:167–9. Ginsburg H, Divo AA, Geary TG, Boland MT, Jensen JB. Effect of mitochondrial inhibitors on intraerythrocytic Plasmodium falciparum in in vitro cultures. J Protozool 1986;33:121 – 5. Krungkrai J, Cerami A, Henderson GB. Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei. Biochemistry 1991;30:1934 – 9. Krungkrai J, Krungkrai SR, Suraveratum N, Prapunwattana P. Mitochondrial ubiquinol-cytochrome c reductase and cytochrome c oxidase: chemotherapeutic targets in malarial parasites. Biochem Mol Biol Inter 1997;42:1007– 14. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193:673–5. Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 1979;65:418 – 20.
.
[22] Krungkrai J, Cerami A, Henderson GB. Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata. Biochemistry 1990;29:6270 – 5. [23] Robinson J, Cooper JM. Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Anal Biochem 1970;33:390 – 9. [24] Krungkrai J, Krungkrai SR, Phakanont K. Antimalarial activity of orotate analogs that inhibit dihydroorotase and dihydroorotate dehydrogenase. Biochem Pharmacol 1992;43:1295 – 301. [25] Rickwood D, Wilson MT, Darley-Usmar VM. Isolation and characteristics of intact mitochondria. In: Darley-Usmar VM, Rickwood D, Wilson MT, editors. Mitochondria: A Practical Approach. Oxford: IRL Press, 1987:1 – 16. [26] Bradford MM. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 – 54. [27] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5. [28] Vinogradov AD, Gavrikova EV, Zuevsky VV. Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. Eur J Biochem 1976;63:365 – 71. [29] Hatefi Y, Galante YM. Isolation of cytochrome b560 from complex II (succinate:ubiquinone oxidoreductase) and its reconstitution with succinate dehydrogenase. J Biol Chem 1980;255:5530– 7. [30] Yu L, Xu JX, Haley PE, Yu CA. Properties of bovine heart mitochondrial cytochrome b560. J Biol Chem 1987;262:1137– 43. [31] Hatefi Y. Resolution of complex II and isolation of succinate dehydrogenase (EC 1.3.99.1). Methods Enzymol 1978;53:27 – 35. [32] LeBlanc SB, Wilson CM. The dihydroorotate dehydrogenase gene homologue of Plasmodium falciparum. Mol Biochem Parasitol 1993;60:349 – 52.
Purification and characterization of Plasmodium falciparum succinate dehydrogenase Nongluk Suraveratum a,1, Sudaratana R. Krungkrai b, Preecha Leangaramgul a, Phisit Prapunwattana a, Jerapan Krungkrai a,* a
Department of Biochemistry, Faculty of Medicine, Chulalongkorn Uni6ersity, Rama IV Road, Bangkok 10330, Thailand b Department of Biochemistry, Faculty of Science, Rangsit Uni6ersity, Patumthani 12000, Thailand Received 2 August 1999; received in revised form 28 September 1999; accepted 28 September 1999
Abstract 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 909 8 kDa and contained two major subunits with molecular masses of 55 9 6 and 3594 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 mM, and kcat 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 Ki values of 81, 13 and 12 mM, 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 mM. The quinone had antimalarial activity against the in vitro growth of P. falciparum with a 50% inhibitory concentration of 0.27 mM and was found to completely inhibit oxygen uptake of the parasite at a concentration of 0.88 mM. 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 mM. Many properties observed in the malarial SDH were found to be different from the host mammalian enzyme. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Plasmodium falciparum; Succinate dehydrogenase; Mitochondrial electron transport complex; Chemotherapeutic target; Antimalarial agent
Abbre6iations: CoQ, 2,3-dimethoxy-5-methyl-1,4-benzoquinone; ETS, electron transport system; Fp, flavoprotein; Ip, iron – sulfur protein; PMSF, phenylmethylsulfonyl fluoride; SDH, succinate dehydrogenase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TTFA, 2-thenoyltrifluoroacetone. * Corresponding author. Tel.: +66-2-252-4986; fax: + 66-2-256-4482. E-mail address: [email protected] (J. Krungkrai) 1 Present address: Unit of Biochemistry, Rajavithi Hospital, Rajavithi Road, Bangkok 10400, Thailand. 0166-6851/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 9 ) 0 0 1 8 0 - 2
216
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
1. Introduction Succinate dehydrogenase (complex II, succinate: ubiquinone oxidoreductase, EC 1.3.99.1) is a tricarboxylic acid (Krebs) cycle enzyme that functions as a primary dehydrogenase, donating electrons to the aerobic and energy-generating respiratory chain in eukaryotic mitochondria and numerous prokaryotes [1]. The enzyme has been isolated and characterized from a number of prokaryotic [2–4] and eukaryotic sources [5,6]. Relatively little is known about the enzyme in the genus Plasmodium. During the asexual blood stage of the malarial parasites, they grow and mature within the erythrocytes of their hosts. The absence of cristate structure in mitochondrial organelle at this stage of the parasite has been demonstrated [7 – 10]. Energy requirement during this stage is provided by metabolizing glucose primarily by anaerobic glycolysis [11,12]. Only two Krebs cycle enzymes, malate dehydrogenase and isocitrate dehydrogenase, have been demonstrated in Plasmodium falciparum and Plasmodium knowlesi [13 – 15]. It has been shown that P. falciparum lack at least some of Krebs cycle enzymes, e.g. a-ketoglutarate dehydrogenase [16]. It has been clearly evident that the malaria parasites have mitochondrial electron transport system (ETS) and oxygen-requiring system that is necessary for growth and survival [6,8,11,12,17,18]. Recently the ETS complex III (ubiquinol-cytochrome c reductase) and complex IV (cytochrome c oxidase) have been purified and characterized in Plasmodium berghei and P. falciparum [9,19]. In this study, the succinate dehydrogenase (SDH) enzyme has been purified from the asexual blood stage, mainly trophozoite, of P. falciparum grown in vitro. Physical and kinetic properties of the purified enzyme are investigated. These properties observed in the malarial SDH are different from those in the mammalian mitochondrial enzyme.
2. Materials and methods
2.1. Culti6ation of malaria parasite
P. falciparum (a pyrimethamine resistant T9 line) was cultivated by a modification of the candle jar method of Trager and Jensen [20], using a 5% hematocrit of human erythrocytes type O suspended in RPMI 1640 medium supplemented with 25 mM Hepes, 32 mM NaHCO3 and 10% fresh human serum type O. The cultures started at low parasitemia ( 1–2%) were changed with the medium twice daily until the cultures had 30% parasitemia and then harvested for enzyme preparation. Synchrony of the culture was maintained by sorbitol procedure of Lambros and Vanderberg [21].
2.2. Purification of enzyme on fast protein liquid chromatographic system The parasites freed from the infected erythrocytes containing mostly trophozoite stage were washed and lysed according to the established procedure [22].The parasite homogenate containing 0.2% octyl glucoside was dialyzed, concentrated and loaded onto a Pharmacia Mono Q FPLC column equilibrated with 5 mM phosphate buffer, pH 8.0, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM EDTA. The column was then washed with the equilibrating buffer and proteins were eluted with a linear gradient from 0 to 600 mM KCl over 40 min at a flow rate of 1 ml min − 1. The 1.0-ml fractions were collected and then measured for SDH enzyme activity. The active fractions from the Mono Q column eluted at 70 mM KCl were dialyzed, concentrated and then loaded onto a Mono S cation-exchange FPLC column which was equilibrated with 5 mM phosphate buffer, pH 6.0, containing 1 mM PMSF and 5 mM EDTA. The column was washed with the same buffer and then eluted with linear gradient of the phosphate buffer from pH 6.0 to pH 8.0 at a flow rate of 1 ml min − 1. The eluates were collected into 38 fractions, each 1.0 ml fraction was determined for SDH activity and protein concentration. The eluates containing the enzyme activity from the Mono S column (at pH 6.3) were pooled,
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
concentrated and then applied to a Superose 6 gel filtration FPLC column equilibrated with 5 mM phosphate buffer, pH 8.0, containing 1 mM PMSF, 5 mM EDTA and 150 mM KCl. The enzyme was eluted with this buffer over 45 min at a flow rate of 0.5 ml min − 1, the 0.5-ml fractions were collected and assayed for the SDH activity. The active fractions were pooled and stored as aliquots at − 196°C. The Superose 6 column was calibrated with the following Pharmacia molecular mass markers: thyroglobulin (670 kDa), immunoglobulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa).
2.3. Enzyme assay and kinetic studies SDH activity was measured by following reduction of cytochrome c at 550 nm and 37°C using a Shimadzu 1601 spectrophotometer equipped with a temperature-controlled unit. The enzymatic reaction, in a total volume of 1.0 ml, contained 50 mM phosphate buffer, pH 8.0, 1 mM succinate, 0.01 mM 2,3-dimethoxy-5-methyl-1,4-benzoquinone (CoQ) and 0.1 mM cytochrome c. A specific inhibitor of SDH, 10 mM malonate, was also included in the enzymatic reaction for correcting non-specific dehydrogenase(s) activities in the parasite homogenate before purification steps. For kinetic studies, the SDH activity on the purified enzyme was measured with dichlorophenolindophenol as a terminal electron acceptor, the absorbance change at 600 nm was then monitored. Kinetic constants, Km and kcat, were determined by fitting data to the Michaelis – Menten equation using non-linear regression of Enzfitter program (Elsevier Biosoft). Inhibitor constants (Ki) were determined from Dixon’s plots. I50 was defined as the concentration of compound having 50% inhibitory effect against the purified enzyme.
2.4. Measurement of oxygen uptake by host cell-free P. falciparum The rate of oxygen uptake for a consistent number of host cell-free P. falciparum parasites was measured in a modified medium containing 75 mM sucrose, 225 mM mannitol, 5 mM MgCl2,
217
5 mM KH2PO4, 1 mM EGTA, 5 mM Hepes, pH 7.4 [9] by using a Clark-type oxygen electrode and YSI oxygen monitor according to the method of Robinson and Cooper [23]. The oxygen uptake reaction in a total volume of 3 ml was followed and recorded at 37°C with a temperature-controlled circulator.
2.5. In 6itro antimalarial test Antimalarial activity against P. falciparum in vitro was quantified by measuring % parasitemia in a 4-day culture in the presence of the tested compounds at various concentrations [24]. All compounds were tested in triplicate at each concentration used. The 50% inhibitory concentration (IC50) was defined as the concentration of the compound causing 50% inhibition of the parasite growth in a 4-day culture, compared with the compound-free control of the parasite culture.
2.6. Other methods Mammalian mitochondria were prepared from mouse liver as described previously [25]. Protein concentrations were determined by the method of Bradford [26] and using bovine serum albumin as standard. SDS-PAGE was performed on a BioRad minislab gel apparatus with a 5% acrylamide stacking gel and 10% acrylamide running gel in the discontinuous buffer system of Laemmli [27]. Nondenaturing-PAGE was performed by using the modified Laemmli’s method in the absence of SDS and 2-mercaptoethanol in all reagents used, and 10% acrylamide running gel was applied. The gels were stained with Coomassie Blue R and visualized by a Bio-Rad molecular analyst PC software image analysis.
3. Results Using the cytochrome c reduction assay, the SDH activity in the parasite homogenate was approximately 309 4% of total dehydrogenase activity. It was noted that after FPLC steps the SDH activity was completely inhibited in the pres-
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
218
Table 1 Purification of succinate dehydrogenase (SDH) from 3.0-ml of packed host erythrocyte-free Plasmodium falciparum Step
Total protein (mg) Total activity (mU)a
Specific activity (mU mg−1)
Yield (%)
Fold purification
1. 2. 3. 4.
139.56 15.04 3.96 0.08
1.37 5.38 6.17 33.13
100 42.3 12.8 1.4
1 3.9 4.5 24.2
Homogenate Mono Q FPLC Mono S FPLC Superose 6 FPLC a
191.08 80.88 24.42 2.65
The mU of enzyme activity is expressed as nmol min−1 at 37°C.
ence of 10 mM malonate. The results of a typical purification of the malarial SDH enzyme are summarized in Table 1. Purification of the parasite enzyme resulted in relatively low yield of 1% and a 24-fold purification. The parasite enzyme was 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 that of − 20°C. Many attempts have been made to reactivate the parasite enzyme activity, e.g. addition of stabilizers (0.1 mM 2-mercaptoethanol or 10 mM 1,4-dithiothreitol or 1 mM succinate) or flavin coenzymes (0.01 mM of FADH2 or FMN). However, these additives had no effect on the reactivating and also stabilizing the enzyme activity during purification. The purification of the malarial enzyme was hampered by its lability property, however, eight purifications were performed. Using nondenaturing PAGE the purified enzyme (eluted from the Superose 6 gel filtration FPLC column, Table 1) exhibited one major band (Fig. 1A). The purified enzyme was then subjected to SDS-PAGE to determine number and molecular mass of enzyme subunits (Fig. 1B). There were few faint bands observed at positions of 90 and 13 –20 kDa when 20 mg of the purified enzyme was loaded onto the gel (Fig. 1B, lane a). The results obtained in Fig. 1 suggest that the parasite enzyme had two major subunits with apparent molecular masses of 5596 and 359 4 kDa (n = 8). The enzyme had a native molecular mass of 909 8 kDa (n= 8) based on the eluting position on the Superose 6 gel filtration FPLC column (Fig. 2).
Kinetic analyses of the parasite SDH demonstrated that the enzyme required both succinate and CoQ for its maximal activity, and followed Michaelis–Menten kinetics for each substrate. The Km and kcat values for both substrates were determined from the plots of the Enzfitter software program and the results are shown in Table 2. The catalytic efficiency of the malarial enzyme, calculated as kcat/Km, was found to be relatively low (Table 2). By using Dixon’s plot, malonate and oxaloacetate, two analogs of the substrate succinate, were found to be effective competitive
Fig. 1. (A) Nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis of purified Plasmodium falciparum succinate dehydrogenase eluted from the Superose 6 gel filtration FPLC column. The amount of protein loaded onto the gel of lanes a and b was 10 and 20 mg, respectively. The arrow indicates position of a major protein band on the gel. (B) Sodium dodecyl sulfate (SDS)-PAGE analysis of purified P. falciparum succinate dehydrogenase. The amount of protein loaded onto the gel of lanes a and b was 20 and 10 mg, respectively. The bars indicate positions of molecular mass marker proteins: phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonic anhydrase (31.0 kDa), trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa).
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
219
Fig. 2. FPLC chromatogram of Plasmodium falciparum succinate dehydrogenase on the Superose 6 gel filtration column. The arrows show eluting positions of calibrated molecular mass proteins. The symbols used were as follows: , total enzyme activity per 0.5-ml fraction; , amount of protein per 0.5-ml fraction. Table 2 Enzyme kinetic and inhibitory constant parameters of Plasmodium falciparum succinate dehydrogenase (SDH) Compound
Km (mM)
kcat (min−1)
Ki (mM)
kcat/Km (M−1 s−1)
Substrates: Succinate CoQ
3.019 0.05a 0.209 0.03
0.119 0.01 0.069 0.01
– –
600 5000
Inhibitors: Malonate Oxaloacetate Fumarate a
13.02 9 0.49 12.06 9 0.81 80.99 9 3.07
Values are mean 9 S.D., taken from five to six separate experiments of the enzyme preparations.
inhibitors of the parasite enzyme. Fumarate, the enzymatic product, showed an inhibitory effect against the malarial SDH (Table 2). The known mammalian ETS complex II inhibitor 2-thenoyltrifluoroacetone (TTFA) had no inhibitory effect against both the parasite enzyme activity and the oxygen uptake by the host cellfree parasites at concentration of 50 mM. The oxygen uptake by these parasites in the assay system as described in Section 2 were found to be 1.56 90.23 nmol min − 1 mg − 1 (n = 3). The parasite’s oxygen uptake showed relatively resistance to TTFA inhibition, only 10% inhibition was observed at 100 mM, whereas TTFA at this con-
centration was found to inhibit more than 90% of the mouse liver mitochondrial oxygen uptake. Chloroquine (100 mM), artemisinin (5 mM) and atovaquone (5 mM) showed no inhibitory effect against the parasite enzyme activity. Interestingly, the parasite SDH activity was sensitive to inhibition of 5-hydroxy-2-methyl-1,4-naphthoquinone (or plumbagin), a CoQ analog, with an I50 of 5 mM. Furthermore, plumbagin was found to completely inhibit the oxygen uptake by the parasite at a concentration at 0.88 mM (or 165 ng ml − 1). It was also demonstrated that it had antimalarial activity against in vitro growth of P. falciparum with an IC50 of 0.27 mM (or 50.8 ng ml − 1).
220
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
4. Discussion The present findings clearly demonstrate that the malaria parasite P. falciparum possesses significant SDH activity. This is the first report on the purification and characterization of the important enzyme complex II responsible for mitochondrial ETS and Krebs cycle in the parasite. Our attempts on the biochemical purifications were hampered by the markedly labile and low specific activity of the malarial enzyme. It is generally accepted that the active site of the enzyme in various sources contains highly reactive sulfhydryl group that participates in the first step of succinate oxidation [28], the sulfhydryl group may be directly involved in the inactivation resulting in an unstable enzyme. This led one to use high amounts of parasites as the starting material to overcome the extremely low yield and purification. The malarial SDH has been purified to near homogeneity (Fig. 1). It had an apparent molecular mass of 90 kDa (Fig. 2) and contained 2 subunits with molecular masses of 55 kDa (Fp, flavoprotein subunit) and 35 kDa (Ip, iron – sulfur protein subunit). Similar results are also obtained from a rodent parasite P. berghei with Fp subunit 63 kDa and Ip subunit 35 kDa [Krungkrai et al. unpublished results]. However, faint bands at molecular masses of 13 – 20 kDa observed on the SDS-PAGE analysis of the enzyme preparation may be two subunits of low molecular masses of cytochrome b which are 15 – 20 and 13 – 16 kDa and reported in both bacterial and beef heart mitochondrial enzymes respectively [2,29,30]. The cytochrome b subunits of the ETS complex II appear to be essential for the assembly of the Fp and Ip subunits of SDH with the inner membrane of mitochondria [30]. The putative membrane anchoring and electron transferring functions of the two smaller subunits of cytochrome b in the malarial SDH enzyme needs to be further characterized. The molecular mass and the Fp and Ip subunits structure of the malarial enzyme were quite different to the SDH obtained from both Escherichia coli and mitochondrial beef heart. In E. coli the enzyme had a molecular mass of 118 kDa based on the nucleotide sequencing
data calculation, which consisted of Fp subunit 64 kDa, Ip subunit 28 kDa and two smaller subunits 19.5 and 17.5 kDa based on the SDSPAGE analysis [2]. The mitochondrial enzyme of beef heart had a molecular mass of 140 kDa and consisted of Fp subunit 70 kDa, Ip subunit 27 kDa and two cytochrome b subunits 15.5 and 13.5 kDa [5,6,29–31]. The catalytic efficiency of the malarial SDH (kcat/Km 600–5000 M − 1 s − 1) was not much different to another two enzymatic complexes of the parasite mitochondrial ETS, complex III ubiquinol-cytochrome c reductase (kcat/Km 12 000 M − 1 s − 1) and complex IV cytochrome c oxidase (kcat/Km 1300 M − 1 s − 1), which have been recently reported [19]. It is noted that the catalytic efficiency of the parasite enzyme is much lower than those of the bacterial and mammalian enzymes [2,5,6]. Several of the kinetic properties of the malarial enzyme differed from those reported for SDH from several other sources. The Km for succinate of the P. falciparum enzyme is 3 mM which compares with 20 mM measured for the E. coli SDH [2], 41 mM for the P. berghei SDH [Krungkrai et al. unpublished results], and 71 mM for the beef heart mitochondrial enzyme [6]. In addition, the Km for CoQ of the P. falciparum SDH is 0.2 mM which compares with 4.2 mM measured for the E. coli enzyme [2]. The malarial enzyme is also sensitive to the competitive inhibitors which have Ki values of 12 mM for oxaloacetate, 13 mM for malonate and 81 mM for fumarate, the enzymatic product. The Ki values for malonate of the beef heart mitochondrial and E. coli enzymes are 0.25 [6], and 200 mM [2], respectively. The Ki values for oxaloacetate and fumarate of the beef heart mitochondrial enzyme are reported to be 1.5 mM and 1.3 mM, respectively [31]. These Km and Ki values suggest that the active site of the malarial SDH is different from the reported enzymes in various organisms. This difference in active site was supported by a comparison of the classical mammalian mitochondrial enzyme inhibitor TTFA [1] with the enzymes from P. falciparum, E. coli [2], and beef heart mitochondria [6]. In contrast to the mammalian mitochondrial enzyme, the bacterial and malarial enzymes are relatively insensitive to
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222
TTFA inhibition. Furthermore, when the oxygen uptake was measured in the malarial parasite and the mouse liver mitochondria, the parasite’s oxygen uptake was relatively resistant to the TTFA inhibition, as compared to the sensitivity to TTFA inhibition of the liver mitochondria. However, TTFA is reported to have relatively low inhibitory effect against the growth of P. falciparum in in vitro culture [17]. Interestingly, the CoQ analog plumbagin may exert its potent antimalarial effect through inhibition of the parasite SDH enzyme and the oxygen uptake activities. It should be also noted here that the parasite enzyme is not the target for the currently used antimalarial drugs, e.g. chloroquine, artemisinin and atovaquone. More recently, atovaquone is reported to specifically inhibit the parasite ETS complex III ubiquinol-cytochrome c reductase with an I50 of 5 nM [19]. During the progress of this work, Takeo and Kita have submitted the nucleotide sequences encoding both Fp and Ip subunits of P. falciparum SDH at GenBank (accession numbers: D86573 and D86574). The deduced amino acid sequences of both Fp and Ip subunits seem to have also unique properties. The calculated molecular masses of the genes of Fp subunit 69 kDa and Ip subunit 38 kDa were rather bigger than the proteins characterized in this report. It is possible that the parasite mitochondrial proteins synthesized in cytosol containing organelle targeting signal may be cleaved during translocation into mitochondrion, but verification of this will be necessary. Nevertheless, difference in the molecular mass has also been observed in the mitochondrial dihydroorotate dehydrogenase of P. falciparum between the deduced amino acid sequence from the nuclear gene [32] and the purified enzyme [8]. The P. falciparum SDH enzyme is indeed a unique enzyme. First, the parasite enzyme is physically and kinetically different from the enzymes of the mammalian tissues and the bacterial system. Second, the classical mammalian mitochondrial enzyme inhibitor 2-thenoyltrifluoroacetone shows no inhibitory effect on the parasite enzyme. Third, the potent antimalarial plumbagin has strong inhibitory effect on the enzyme. These
221
unique properties suggest that the SDH enzyme of the malarial parasites may be a potential target for selective chemotherapeutic agents.
Acknowledgements We thank Rachanok Kanjanarithisak and Sanya Kudan for their technical assistance with parasite cultivation, animal handling and enzyme purification. A part of this work was the M.S. thesis of N. Suraveratum. This work was supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. J. Krungkrai is grateful for a career development award from the National Science and Technology Development Agency of Thailand.
References [1] Hatefi Y. The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 1985;54:1015 – 69. [2] Kita K, Vibat CRT, Meinhardt S, Guest JR, Gennis RB. One-step purification from Escherichia coli of complex II (succinate:ubiquinone oxidoreductase) associated with succinate reducible cytochrome b556. J Biol Chem 1989;264:2672– 7. [3] Moll R, Schafer G. Purification and characterization of an archaebacterial succinate dehydrogenase complex from the plasma membrane of the thermoacidophile Sulfolobus acidocaldarius. Eur J Biochem 1991;207:593 – 600. [4] Reddy TLP, Weber MM. Solubilization, purification and characterization from membranes of M. phlei. J Bacteriol 1986;167:1 – 6. [5] Hatefi Y, Stiggall DL. Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol 1978;53:21 – 7. [6] Tushurashvili PR, Gavrikova EV, Ledenev AN, Vinogradov AD. Studies on the succinate dehydrogenating system. Isolation and properties of the mitochondrial succinate-ubiquinone reductase. Biochim Biophys Acta 1985;809:145 – 59. [7] Fry M, Beesley JE. Mitochondria of mammalian Plasmodium spp. Parasitology 1991;102:17 – 26. [8] Krungkrai J. Purification, characterization and localization of mitochondrial dihydroorotate dehydrogenase in Plasmodium falciparum, human malaria parasite. Biochim Biophys Acta 1995;1243:351– 60. [9] Krungkrai J, Krungkrai SR, Bhumiratana A. Plasmodium berghei: partial purification and characterization of the
222
[10]
[11]
[12] [13]
[14] [15]
[16]
[17]
[18]
[19]
[20] [21]
N. Sura6eratum et al. / Molecular and Biochemical Parasitology 105 (2000) 215–222 mitochondrial cytochrome c oxidase. Exp Parasitol 1993;77:136 – 46. Langreth SG, Jensen JB, Reese RT, Trager W. Fine structure of human malaria in vitro. J Protozool 1978;25:443 – 52. Scheibel LW. Plasmodial metabolism: carbohydrate. In: Wernsdorfer WH, McGregor I, editors. Malaria, vol. I. New York: Churchill Livingstone, 1988:171–217. Sherman IW. Biochemistry of Plasmodium (malaria parasites). Microbiol Rev 1979;43:453–95. Lang-Unnash N. Purification and properties of Plasmodium falciparum malate dehydrogenase. Mol Biochem Parasitol 1992;50:17 – 26. Lang-Unnash N. Plasmodium falciparum: antiserum to malate dehydrogenase. Exp Parasitol 1993;80:357–9. Sahni SK, Saxena N, Puri SK, Dutta GP, Pandey VC. NADP-specific isocitrate dehydrogenase from the simian malaria parasite Plasmodium knowlesi: partial purification and characterization. J Protozool 1992;39:338–42. Blum JJ, Ginsburg H. Absence of alpha-ketoglutarate dehydrogennase activity and presence of CO2-fixing activity in Plasmodium falciparum grown in vitro in human erythrocytes. J Protozool 1984;34:167–9. Ginsburg H, Divo AA, Geary TG, Boland MT, Jensen JB. Effect of mitochondrial inhibitors on intraerythrocytic Plasmodium falciparum in in vitro cultures. J Protozool 1986;33:121 – 5. Krungkrai J, Cerami A, Henderson GB. Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei. Biochemistry 1991;30:1934 – 9. Krungkrai J, Krungkrai SR, Suraveratum N, Prapunwattana P. Mitochondrial ubiquinol-cytochrome c reductase and cytochrome c oxidase: chemotherapeutic targets in malarial parasites. Biochem Mol Biol Inter 1997;42:1007– 14. Trager W, Jensen JB. Human malaria parasites in continuous culture. Science 1976;193:673–5. Lambros C, Vanderberg JP. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 1979;65:418 – 20.
.
[22] Krungkrai J, Cerami A, Henderson GB. Pyrimidine biosynthesis in parasitic protozoa: purification of a monofunctional dihydroorotase from Plasmodium berghei and Crithidia fasciculata. Biochemistry 1990;29:6270 – 5. [23] Robinson J, Cooper JM. Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Anal Biochem 1970;33:390 – 9. [24] Krungkrai J, Krungkrai SR, Phakanont K. Antimalarial activity of orotate analogs that inhibit dihydroorotase and dihydroorotate dehydrogenase. Biochem Pharmacol 1992;43:1295 – 301. [25] Rickwood D, Wilson MT, Darley-Usmar VM. Isolation and characteristics of intact mitochondria. In: Darley-Usmar VM, Rickwood D, Wilson MT, editors. Mitochondria: A Practical Approach. Oxford: IRL Press, 1987:1 – 16. [26] Bradford MM. A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 – 54. [27] Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680 – 5. [28] Vinogradov AD, Gavrikova EV, Zuevsky VV. Reactivity of the sulfhydryl groups of soluble succinate dehydrogenase. Eur J Biochem 1976;63:365 – 71. [29] Hatefi Y, Galante YM. Isolation of cytochrome b560 from complex II (succinate:ubiquinone oxidoreductase) and its reconstitution with succinate dehydrogenase. J Biol Chem 1980;255:5530– 7. [30] Yu L, Xu JX, Haley PE, Yu CA. Properties of bovine heart mitochondrial cytochrome b560. J Biol Chem 1987;262:1137– 43. [31] Hatefi Y. Resolution of complex II and isolation of succinate dehydrogenase (EC 1.3.99.1). Methods Enzymol 1978;53:27 – 35. [32] LeBlanc SB, Wilson CM. The dihydroorotate dehydrogenase gene homologue of Plasmodium falciparum. Mol Biochem Parasitol 1993;60:349 – 52.