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Trypanosomatid Hydrogen Peroxidase Metabolism View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 221, number 2, 427-431 FEB 05098 September 1987 Trypanosomatid hydrogen peroxidase metabolism P.G. Penketh, W.P.K. Kennedy*, C.L. Patton and A.C. Sartorelli MacArthur Center for Parasitology and Tropical Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA and * Wolfson Molecular Biology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WCLE 7HT, England Received 7 July 1987 The rate of whole cell HZ02 metabolism in several salivarian and stercorarian trypanosomes and Leishrnania species was measured. These cells metabolized Hz02 at rates between 2.3 and 48.2 nmol/lO* cells per min depending upon the species employed. Hz02 metabolism was largely insensitive to NaN3, implying that typi- cal catalase and peroxidase haemoproteins are not important in Hz02 metabolism. The metabolism of H202, however, was almost completely inhibited by N-ethylmaleimide. In representative species, Hz02 metabolism was shown to occur through a trypanothione-dependent mechanism. Hydrogen peroxide; Trypanothione; Macrophage; (Trypanosoma, Leishmania) 1. INTRODUCTION been reported that L. donovani possesses a surface-membrane acid phosphatase which reduces Many trypanosomatids have been reported to the magnitude of the oxidative burst of lack or to be extremely deficient in enzyme systems neutrophils, inferring a pathophysiological role for necessary for the removal of Hz02 (i.e. catalase, this enzyme [5]. However, defense mechanisms glutathione peroxidase) [l-5]. Defense against endogenously generated Hz02 and the mechanisms against H202, however, appear to be possibly diminished quantity of Hz02 produced a ubiquitous requirement of most aerobic cells [6]. during the oxidative burst would still be required. Leishmania and Trypanosoma cruzi Recently, T. brucei has been shown to contain a .trypomastigotes must also withstand the oxidative novel enzymic HzOz-metabolizing system. This killing mechanism of phagocytic macrophages and system utilizes NADPH for the reduction of Hz02 other host cells [7]. This suggests the presence of and has an absolute requirement for the dithiol co- mechanisms other than catalase and glutathione factor trypanothione [N’,M-bis(L-y-glutamyl-L- peroxidase activities to fulfill this vital role. It has hemicysteinylglycyl)spermidine] [9, lo]. Similar ac- tivity was measured when organic hydroperoxides Correspondence address: P.G. Penketh, MacArthur were used as substrates, implying that an addi- Center for Parasitology and Tropical Medicine, Yale tional role exists for this activity in the reduction of University School of Medicine, 333 Cedar Street, New lipid hydroperoxides [lo]. The precise details of Haven, CT 06510, USA the reaction mechanisms involved are not yet clear. A system analogous to the host glutathione reduc- Abbreviations: BSA, bovine serum albumin; NEM, N- ethylmaleimide; GSSWGSH, oxidized and reduced tase/glutathione peroxidase system utilizing glutathione, respectively; NADH, reduced nicotinamide trypanothione was anticipated. adenine dinucleotide; NADP+/NADPH, oxidized and Trypanothione reductase has been purified to reduced nicotinamide adenine dinucleotide phosphate, homogeneity from several trypanosomatides respectively; TWT(SHl2, oxidized and reduced [ 11,121. Trypanothione peroxidase activity has trypanothione, respectively been inferred in crude extracts or crude extracts Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/87/$3.50 0 1987 Federation of European Biochemical Societies 427 Volume 221, number 2 FEBS LETTERS September 1987 supplemented with additional trypanothione amastigotes were obtained and purified from in- reductase by a presumptive coupled enzymic assay fected Syrian hamster spleens as described by [9,10,13]; however, the expected trypanothione Channon et al. [19]. L. braziliensis and L. mex- peroxidase activity has so far eluted detection by icana amazonensis promastigotes were cultured in direct assay in both T. brucei and T. cruzi Schneider’s medium containing 2 mM glutamine, [9,10,14]. The following reaction scheme has been 10% fetal calf serum, 100 U/ml of penicillin and proposed for the metabolism of hydroperoxides 100 pg/ml of streptomycin at 25°C. L. braziliensis [13]. amastigotes were grown in culture as described by Pan [20]. L. tarantolae was cultured in LT medium ROOH ROH i- Hz0 at 27°C. All cells were washed in incubation buffer (5 mM KCl, 80 mM NaCl, 2 mM MgC12, 16.2 mM Na2HP04, 3.8 mM NaHzP04, 50 mM D-glucose, 0.27 mM phenol red and 1.5 mg/ml of BSA, ad- $r y panot hione per oxid justed to pH 7.4) and stored on ice prior to use. Hz02 metabolism of cell suspensions was measured as described in the legend to table 1. NADPH dependent Hz02 consumption was measured as follows. Trypanosomes were washed in glucose-free incubation buffer, incubated for 10 min at room temperature, pelleted by cen- trifugation (3000 x g for 10 min) and osmotically lysed by suspension in distilled water at a density equivalent to lo9 cells/ml. This suspension was NADP+ NADPH + H+ centrifuged at 20000 x g for 10 min and 1 vol. of 1 M Tris-HCl (pH 8.0) containing 10 mM EDTA Here, we have measured the ability of 12 buffer was added to 9 vols of the supernatant. trypanosomatids to metabolize H202. NADPH and NADP+ were added to give final concentrations of 100 PM (the inclusion of 100 FM 2. MATERIALS AND METHODS NADP+ corrects for any residual NADP+ reduc- ing capacity). Hz02 was then added to the sample Enzymes and co-factors were obtained from cuvette at concentrations between 2.5 and 20,uM Sigma. Synthetic trypanothione was a kind gift and the change in absorbance at 340 nm was from Dr A. Fairlamb. All other chemicals were of followed at 37°C against an equivalent sample AR grade. Incubation buffer was composed as in without H202. This procedure was further [lOI. modified in the case of T. lewisi because of the Bloodstream trypomastigotes of T. brucei high NADP+ reducing capacity of these lysates. rhodesiense (YTat 1. I), T. equiperdum, T. evansi These cells were lysed at a level of 5 x 10’ cells/ml and T. vivax were prepared and purified as and 250 PM NADP+ was used instead of 100 PM described by Lanham [15]. T. lewisi and T. con- NADPH and 1OOpM NADP+. Hz02 was then golense were prepared in a similar manner, except added after the mixture had been incubated for that lethally irradiated rats were used as hosts. 5 min. L. mexicana amazonensis promastigotes Procyclic forms of T. brucei rhodesiense were were treated in a manner analogous to that of T. cultured in Cunningham’s medium supplemented lewisi except that these cells, due to their resistance with 10% fetal calf serum at 27°C. T. cruzi Y to osmotic lysis, were lysed at a level of lo9 strain and X 10 strain epimastigotes were cultured cells/ml by nitrogen cavitation in distilled water. as described by Widmer [ 161. T. cruzi In all of these preparations, the intrinsic trypomastigotes (Y strain) were prepared ac- trypanothione reductase activity greatly exceeded cording to Piras et al. [17]. L. donovani promas- that of the peroxidase; for example, a 50-fold dif- tigotes were cultured and isolated as described by ference in activity has been reported in T. brucei Neal [ 181 for L. tropica major. L. donovani [13]. The HzOz-mediated oxidation of NADPH 428 Volume 221, number 2 FEBS LETTERS September 1987 Table 1 were prepared using an Amicon PM-10 filter The initial rate of Hz02 metabolism by various (nominal molecular mass cut off at 10 kDa). The trypanosome and Leishmaniu species high molecular mass fraction was washed with 2-times the initial lysate volume of distilled Hz0 Organism nmol Hz02 con- and suspended in a small volume of H20. Catalase sumed per 10’ cells activity was measured using an 02 electrode as in during the 1st min [91. of exposure to 20rM Hz02 (*SD) 3. RESULTS AND DISCUSSION Salivarian trypanosomes All of the cell lines studied were able to T. brucei rhodesiense metabolize Hz02 (table 1). The high consutiption trypomastigotes 3.5 k 0.6 (5) of Hz02 observed in T. cruzi trypomastigotes com- T. brucei rhodesiense pared to epimastigote forms is consistent with their procyclic 6.4 f 1.2 (5) greater resistance to the phagocytic killing T. equiperdum trypomastigotes mechanism and to oxidant stress in the form of Lump 1559 3.7 -t 0.5 (4) Hz02 [21]. The various forms of L. donovani, L. T. evansi SN trypomastigotes mexicana, L. braziliensis, T. cruzi and T. lewisi Lump 1559 3.5 Ik 0.3 (5) studied showed a progressive decrease in the rate T. evansi SAK trypomastigotes 4.2 f 0.3 (5) T. vivax trypomastigotes 3.5 f 0.2 (3) of Hz02 consumption at relatively low (< 10 PM) T. congolense trypomastigotes concentrations of H202, whereas the salivarian TREU 1290 3.6 + 0.4 (3) trypanosomes gave more linear kinetics (not shown). Hz02 metabolism was largely insensitive Storcorarian trypanosomes to 1 mM NaN3, except for L. donovani T. lewisi trypomastigotes 19.6 + 3.0 (7) amastigotes, which implied that typical T. cruzi trypomastigotes Y strain 48.2 + 1.9 (3) catalase/peroxidase haemoproteins are not impor- 18.3 k 1.5 (3) T. cruzi epimastigotes Y strain tant in the metabolism of H202. Hz02 metabolism, T. cruzi epimastigotes however, was inhibited 85-100% by pretreatment X 10 strain 4.2 f 0.2 (3) with NEM (table 2). L. donovani amastigotes Leishmania behaved differently, in that a 40% inhibition of the L. donovani promastigotes 4.3 f 0.2 (3) initial rate of Hz02 metabolism was produced by L. donovani amastigotes 2.3 k 0.2 (3) 1 mM NaN3, suggesting the presence of a catalase- L. brasiliensis promastigotes 8.3 f 0.3 (3) type activity.
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