Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Extramitochondrial localization of NADH-fumarate reductase in trypanosomatids
Introduction
Trypanosomatids are parasitic protozoa causing several diseases including Chagas’ disease, sleeping sickness and a variety of leishmaniases in humans, as well as nagana in cattle. Intermediate metabolism in these parasites shows substantial differences with their mammalian counterpart, not only when comparing species but also when comparing different stages within a given species (Boveris et al., 1986, Clarkson et al., 1989, Chen et al., 2001, Denicola-Seoane et al., 1992, Fairlamb and Opperdoes, 1986, Hill, 1976, Santhamma and Bhaduri, 1995, Turrens, 1989).
Several studies have shown that a mitochondrial respiratory chain of trypanosomatids utilizes significant amounts of succinate as the primary electron donor (Turrens, 1989, Denicola-Seoane et al., 1992). In addition, trypanosomes excrete succinate to the extracellular environment, particularly under anaerobic conditions, suggesting that this metabolite may become a terminal sink of reducing equivalents (Flynn and Bowman, 1973, Cazzulo, 1992). Given this dual role of succinate in trypanosomatids we have proposed that interfering with succinate production should be lethal to these cells (Turrens et al., 1999, Turrens, 1999). What makes this hypothesis more attractive is that one of the succinate-producing enzymes, NADH-fumarate reductase, is absent in mammalian cells but has been identified in trypanosomes and various species of Leishmania and Plasmodium sp. (Boveris et al., 1986, Chen et al., 2001, Fry and Beesley, 1991). Thus, this enzyme may provide a unique target against the diseases caused by these parasites.
Since the enzyme NADH-fumarate reductase was first identified in trypanosomatids, It has been generally accepted that was confined to the mitochondrial matrix (Klein et al., 1975). Several reasons led to this assumption. First, the enzyme reverts a step in the Krebs cycle, and generates succinate, which is a mitochondrial substrate. Second, although this enzyme is not an integral protein this enzyme is mostly membrane bound (Mracek et al., 1991, Christmas and Turrens, 2000) and therefore its activity is in part associated with mitochondrial markers, which contaminate all membrane fractions (Boveris et al., 1986). This study was designed to determine whether fumarate reductase is confined to the mitochondrial matrix or is present in other intracellular compartments. The results show that in three different species of trypanosomatids T. brucei procyclic trypomastigotes, T. cruzi epimastigotes (Tulahuen and Y strains) and L. donovani promastigotes (S-2 strain) the enzyme NADH-fumarate reductase is located, at least in part, in an extramitochondrial compartment.
Section snippets
Cell cultures and processing
Trypanosoma brucei procyclic trypomastigotes were grown at 28 °C, in SDM-79 medium JRH Biosciences, Lenexa, KS, supplemented with 10% heat-inactivated fetal calf serum and penicillin-streptomycin 50 units/ml. The cells were harvested in a buffer containing 0.23 M mannitol, 0.07 M sucrose, 0.1 mM EDTA and 10 mM Tris–HCl (pH 7.4) buffer A.
T. cruzi epimastigotes (Y and Tulahuen strains) were grown at 28 °C in a liquid medium consisting of brain-heart infusion 33 g/l, disodium hydrogen phosphate 4
Results
The specific activity of NADH-fumarate reductase in homogenates of T. brucei procyclic trypomastigotes was 82±9 munits/mg protein (n=13) while in the Tulahuen and Y strains of T. cruzi epimastigotes the specific activities were 17±5 (n=15) and 15±3 munits/mg protein (n=4), respectively.
In order to estimate the intracellular localization of fumarate reductase, T. brucei procyclic trypomastigotes and T. cruzi epimastigotes were permeabilized with digitonin as described in Section 2. The
Discussion
The results shown in this report indicate that when either T. brucei procyclic trypomastigotes, T. cruzi epimastigotes or L. donovani promastigotes were permeabilized with digitonin, the enzyme NADH-fumarate reductase is released at lower digitonin concentrations than those required to permeate the inner mitochondrial membrane. Our experiments do not rule out that a proportion of fumarate reductase may be located in the mitochondrial matrix, since the release of fumarate reductase has not
Acknowledgements
This work was supported by a PHS grants # R15 AI/32703-01 and R15 AI/OD39692-01to J.F.T. The authors thank Dr Roberto Docampo for his helpful comments and for his help with the experiments involving L. donovani.
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