Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi

Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi

FEMS Microbiology Letters 183 (2000) 225^228 www.fems-microbiology.org Separation of NADH-fumarate reductase and succinate dehydrogenase activities ...

167KB Sizes 0 Downloads 16 Views

FEMS Microbiology Letters 183 (2000) 225^228

www.fems-microbiology.org

Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi Patrick B. Christmas, Julio F. Turrens * Department of Biomedical Sciences, College of Allied Health Professions, UCOM 6000, University of South Alabama, Mobile, AL 36688, USA Received 18 November 1999; received in revised form 10 December 1999; accepted 14 December 1999

Abstract A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265^271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Trypanosoma cruzi; NADH-fumarate reductase ; Succinate dehydrogenase ; Electron transfer; Mitochondria ; Metabolism

1. Introduction The enzyme NADH-fumarate reductase catalyzes the reduction of fumarate, generating succinate as a product. This enzyme is absent from mammalian cells but it has been found in prokaryotic cells [1^3], in protozoal parasites of the genera Trypanosoma [4^6], Plasmodium [7], Leishmania [5,6] and in helminths [8,9]. The physiological role of this enzyme in most species is to provide a route for elimination of excess reduction equivalents under anaerobic conditions. Although since 1962 several groups have shown that trypanosomes accumulate succinate under anaerobic conditions [10^13], two recent reviews have questioned the physiological role of fumarate reductase in trypanosomatids [14,15]. In these reports, Tielens and Van Hellemond indicate that trypanosomes do not accumulate succinate during anaerobiosis and proposed that the NADH-fumarate reductase activity might be actually an artifact, probably catalyzed by either succinate dehydrogenase working in reverse or by some other dehydrogenases [14,15]. The authors argue that this enzyme cannot exist in trypanosomatids because fumarate reductases require a speci¢c lowpotential quinone not found in trypanosomatids [14]. This argument is debatable since many oxidoreductases are

very e¤cient without any prosthetic group (i.e. lactic dehydrogenase, malate dehydrogenase). Moreover, NADHfumarate reductase catalyzes a reaction with an vE³P that is 210 mV more positive (therefore more favorable) than the vE³P for the NADH-dependent reduction of pyruvate catalyzed by lactic dehydrogenase. The results reported in this communication show that succinate dehydrogenase and fumarate reductase activities can be separated, and remain fully functional. Moreover, we present additional evidence that, although partially puri¢ed forms of NADH-fumarate reductase transfer electrons to other acceptors, fumarate inhibits these reactions suggesting that it is the preferred substrate. 2. Materials and methods 2.1. Cell cultures Trypanosoma cruzi epimastigotes (Y strain) were grown at 28³C in a liquid medium consisting of brain-heart infusion (37 g l31 ), hemin chlorohydrate (20 mg l31 dissolved in 50% triethanolamine), penicillin-streptomycin (50 U ml31 ) and 10% heat-inactivated newborn calf serum [16]. 2.2. Membrane treatment

* Corresponding author. Tel. : +1 (334) 380-2714; Fax: +1 (334) 380-2714; E-mail: [email protected]

The cells were collected in MST bu¡er (0.23 M manni-

0378-1097 / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 6 4 6 - 1

FEMSLE 9218 3-2-00

226

P.B. Christmas, J.F. Turrens / FEMS Microbiology Letters 183 (2000) 225^228

tol, 0.07 M sucrose, 5 mM Tris^HCl, pH 7.4) containing several protease inhibitors (10 Wg ml31 leupeptin, 10 Wg ml31 bestatin and 10 Wg ml31 pepstatin). The cell suspension was then supplemented with 0.2% Triton X-100 and homogenized using a Potter-Elvehjem Te£on-glass homogenizer to disrupt the cell membrane. The membrane fraction was centrifuged at 105 000Ug for 35 min. The supernatant (containing the cytosolic fraction) was discarded. The membrane fraction (M0 ) was assayed for both succinate dehydrogenase and fumarate reductase activity and was then supplemented with 300 mM KCl followed by agitation in a vortex to extract fumarate reductase. After 30 min on ice, the membrane fraction was centrifuged again. The pellet was resuspended in MST bu¡er and both the new membrane fraction (M1 ) and the supernatant (S1 ) assayed for both activities. NADH-fumarate reductase was measured spectrophotometrically as the fumarate-dependent rate of NADH oxidation at 340 nm (O = 6.22 mM31 cm31 ) using 250 WM NADH and 0.5 mM fumarate. Succinate dehydrogenase was measured as described elsewhere [17]. NADH-fumarate reductase was partially puri¢ed as previously described [18]. The measurements were carried out in a bu¡er containing 125 mM KCl, 30 mM HEPES (pH 7.0) at 30³C. Hydrogen peroxide formation was measured as the increase in £uorescence of p-hydroxy-phenylacetic acid in the presence of horseradish peroxidase in a Hitachi model F-2000 spectrophoto£uorometer as previously described [19]. All other activities were determined spectrophotometrically using a Beckman DU-65 spectrophotometer. Superoxide formation was estimated by monitoring epinephrine (1 mM) oxidation and its inhibition by superoxide dismutase at 480 nm as described in [20]. The reduction rates of either cytochrome c (20 WM) or nitro blue tetrazolium (0.1 mM) were monitored at 550 nm and 490 nm, respectively [21]. 2.3. Reagents Most reagents (dichlorophenol indophenol, epinephrine, fumarate, NADH, phenazine methosulfate, nitro blue tetrazolium, succinate), proteins (cytochrome c, horseradish peroxidase) and bu¡er components (KCl, sucrose, mannitol, HEPES, Tris^HCl, etc.) were purchased from Sigma Chemical Company (St. Louis, MO, USA). Superoxide dismutase (Peroxinorm0 ) was obtained from Gru«nenthal GmbH, Stolberg, Germany. 3. Results and discussion 3.1. Fumarate reductase and succinate dehydrogenase activities are di¡erent enzymes In 1975, Klein et al. proposed that the enzyme NADH-

Fig. 1. NADH-fumarate reductase (FR) and succinate dehydrogenase (SDH) in several fractions isolated from T. cruzi epimastigotes. The fraction named M0 corresponds to the original membrane preparation; M1 corresponds to the membrane fraction isolated after supplementing the original membrane fraction with high salt (300 mM KCl) ; S1 corresponds to the 105 000Ug supernatant from the incubation with high salt.

fumarate reductase in trypanosomes was membrane bound [11]. Experiments by our group, however, showed that the enzyme NADH-fumarate reductase could be released from the membrane by incubation with 300 mM KCl [22] and be puri¢ed further by precipitation between 30% and 40% ammonium sulfate [18]. In order to demonstrate that succinate dehydrogenase and fumarate reductase are, indeed, two independent proteins, we measured both activities in the membrane fraction of T. cruzi epimastigotes and in the soluble fraction resulting from incubation with high salt. Succinate dehydrogenase (Complex II) is a constitutive enzyme in the mitochondrial membrane. This activity remains associated with the membrane fraction, even after treatment with 300 mM KCl. Fig. 1 shows that, although both activities are detectable in the particulate fraction isolated from T. cruzi homogenates (M0 ), an increase in ionic strength causes NADH-fumarate dehydrogenase to be released into the supernatant while succinate dehydrogenase remains associated with the particulate fraction. Moreover, not only there was no substantial change in speci¢c activity for either fraction, but also the fraction retaining succinate dehydrogenase activity (M1 ) is almost completely depleted of NADH-fumarate reductase and vice versa. 3.2. Speci¢city of fumarate as the electron acceptor for fumarate reductase There are many oxidoreductases which physiologically use a single electron acceptor or transfer electrons to a

FEMSLE 9218 3-2-00

P.B. Christmas, J.F. Turrens / FEMS Microbiology Letters 183 (2000) 225^228

227

Table 1 Inhibitory e¡ect of fumarate (1 mM) on NADH-dependent reduction of several acceptors Reagent

Inhibition by fumarate (%)

Inhibition by superoxide dismutase (%)

Nitro blue tetrazolium Epinephrine Oxygen (measured as H2 O2 ) Cytochrome c

65 77 84 75

74 100 N/Aa 70

The inhibitory e¡ect by superoxide dismutase suggests that superoxide anion is an intermediate in these processes. The results are expressed as percent inhibition relative to the reduction rate of every acceptor in the absence of fumarate. Each value corresponds to the average of two to ¢ve experiments with di¡erent preparations of NADH-fumarate reductase. a N/A, not assayed since H2 O2 is the product of the spontaneous dismutation of superoxide anion and, therefore, not inhibitable by superoxide dismutase.

variety of acceptors, including nitro blue tetrazolium salts, phenazine methosulfate, dichlorophenol indophenol or cytochrome c (i.e. £avoproteins such as NADH dehydrogenase and succinate dehydrogenase). NADH-fumarate reductase is not an exception. Results from our laboratory have shown that the enzyme also transfers electrons to other acceptors (including cytochrome c and oxygen) but is unable to transfer electrons to maleic acid, the transisomer of fumarate [22,23]. This suggests that the active site of the enzyme recognizes fumarate but not its transisomer. Superoxide formation by fumarate reductase was also reported in Escherichia coli [24]. Table 1 shows that the NADH-dependent reduction of several electron acceptors is inhibited by addition of fumarate. Superoxide formation was determined using epinephrine, since this probe is oxidized rather than reduced by superoxide anion. We also used nitro blue tetrazolium and cytochrome c, but these probes may be reduced by either superoxide anion or through direct electron transfer at the active site of several reductases. The total inhibition of epinephrine oxidation, and the incomplete inhibition (70^75%) of the reduction of either cytochrome c or nitro blue tetrazolium by superoxide dismutase, indicate that these processes were mostly mediated by superoxide anion. Since hydrogen peroxide formation results from either the divalent reduction of one oxygen molecule or from the spontaneous dismutation of superoxide anion, superoxide dismutase was not tested in this assay. Fumarate also inhibited all these processes by 65^80% suggesting that when the physiological substrate is present, these side reactions are virtually stopped. The incomplete inhibition by fumarate might result from additional NADH-dependent oxidoreductases present in these preparations (i.e. £avoproteins) or simply because some of the electrons in the active site might still be transferred to stronger oxidants (i.e. cytochrome c, oxygen). In summary, the results presented in this communication indicate that NADH-fumarate reductase and succinate dehydrogenase are separate enzymes. Although in the absence of fumarate the enzyme might transfer electrons to alternative substrates, fumarate appears to be the preferred acceptor.

Acknowledgements This work was supported by a PHS grant #R15AI39692-01 (to J.F.T.). The authors want to express their gratitude to Mr. Rafael Hernandez, Mr. Demetrius Robinson and Mr. Benjamin P. Watts, for their technical assistance. P.B.C. and D.R. were supported by a PHS grant #2R25GM49005. References [1] Dickie, P. and Weiner, J.H. (1979) Puri¢cation and characterization of membrane bound fumarate reductase for anaerobically grown Escherichia coli. Can. J. Biochem. 57, 813^1121. [2] Mendz, G.L., Hazell, S.L. and Srinivasan, S. (1995) Fumarate reductase: a target for therapeutic intervention against Helicobacter pylori. Arch. Biochem. Biophys. 321, 153^159. [3] Schro«der, I., Gunsalus, R.P., Ackrell, B.A.C., Cochran, B. and Cecchini, G. (1991) Identi¢cation of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis. J. Biol. Chem. 266, 13572^13579. [4] Boveris, A., Hertig, C.M. and Turrens, J.F. (1986) Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi. Mol. Biochem. Parasitol. 19, 163^169. [5] Santhamma, K.R. and Bhaduri, A. (1995) Characterization of the respiratory chain of Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 75, 43^53. [6] Turrens, J.F., Rubbo, H., Denicola-Seoane, A., Moreno, S.N.J. and Docampo, R. (1992) Fumarate reductase activity in Trypanosoma cruzi epimastigotes and amastigotes and Leishmania donovani promastigotes. Am. J. Trop. Med. Hyg. 47, Abstr. 75. [7] Fry, M. and Beesley, J.E. (1991) Mitochondria of mammalian Plasmodiun spp.. Parasitology 102, 17^26. [8] Hata-Tanaka, A., Kita, K., Furushima, R., Oya, H. and Itoh, S. (1988) ESR studies on iron-sulfur clusters of complex II in Ascaris suum mitochondria which exhibits strong fumarate reductase activity. FEBS Lett. 242, 183^186. [9] Prichard, R.K. (1973) The fumarate reductase reaction of Haemonchus contortus and the mode of action of some anthelmintics. Int. J. Parasitol. 3, 409^417. [10] Cazzulo, J.J. (1992) Aerobic fermentation of glucose by trypanosomatids. FASEB J. 6, 3153^3161. [11] Klein, R.A., Linstead, D.J. and Wheeler, M.V. (1975) Carbon dioxide ¢xation in Trypansomatids. Parasitology 71, 93^107. [12] Ryley, J.F. (1962) Studies on the metabolism of the protozoa. 9. Comparative metabolism of blood-stream and culture forms of Trypanosoma rhodesiense. Biochem. J. 85, 211^223.

FEMSLE 9218 3-2-00

228

P.B. Christmas, J.F. Turrens / FEMS Microbiology Letters 183 (2000) 225^228

[13] Srivastava, H.K. and Bowman, I.B.R. (1972) Adaptation in oxidative metabolism of Trypanosoma rhodesiense during transformation in culture. Comp. Biochem. Physiol. 40, 973^981. [14] Tielens, A.G.M. and Van Hellemond, J.J. (1998) Di¡erences in energy metabolism between Trypanosomatidae. Parasitol. Today 14, 265^271. [15] Tielens, A.G.M. and Van Hellemond, J.J. (1999) More di¡erences in energy metabolism between trypanosomatidae - Reply. Parasitol. Today 15, 347^348. [16] Warren, L. (1960) Metabolism of Schizotrypanum cruzi Chagas. I. E¡ect of culture age and substrate concentration on respiratory rate. J. Parasitol. 46, 529^539. [17] Denicola-Seoane, A., Rubbo, H., Prodanov, E. and Turrens, J.F. (1992) Succinate-dependent metabolism in Trypanosoma cruzi epimastigotes. Mol. Biochem. Parasitol. 54, 43^50. [18] Turrens, J.F., Watts Jr., B.P., Zhong, L. and Docampo, R. (1996) Inhibition of Trypanosoma cruzi and T-brucei NADH fumarate reductase by benzindazole and anthelmintic imidazole derivatives. Mol. Biochem. Parasitol. 82, 125^129. [19] Hyslop, P.A. and Sklar, L.A. (1984) A quantitative £uorimetric assay

[20]

[21]

[22]

[23]

[24]

for the determination of oxidant production by polymorphonuclear leukocytes: its use in simultaneous £uorimetric assay of cellular activation processes. Anal. Biochem. 141, 280^286. Misra, H.P. and Fridovich, I. (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170^3175. Picker, S.D. and Fridovich, I. (1984) On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium. Arch. Biochem. Biophys. 228, 155^158. Mracek, J., Snyder, S.J., Chavez, U.B. and Turrens, J.F. (1991) A soluble fumarate reductase in Trypanosoma brucei procyclic trypomastigotes. J. Protozool. 38, 554^558. Turrens, J.F. (1987) Possible role of the NADH-fumarate reductase in superoxide anion and hydrogen peroxide production in Trypanosoma brucei. Mol. Biochem. Parasitol. 25, 55^60. Imlay, J.A. (1995) A metabolic enzyme that rapidly produces superoxide, fumarate reductase of Escherichia coli. J. Biol. Chem. 270, 19767^19777.

FEMSLE 9218 3-2-00