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Characterization of an ecto-ATPase of Tritrichomonas foetus
Veterinary Parasitology 103 (2002) 29–42
Characterization of an ecto-ATPase of Tritrichomonas foetus José B. Jesus a,b , Angela H.C.S. Lopes b , José R. Meyer-Fernandes a,∗ a
Departamento de Bioqu´ımica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21541-590 Rio de Janeiro, RJ, Brazil b Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio de Janeiro, RJ, Brazil Received 1 June 2001; received in revised form 29 August 2001; accepted 29 August 2001
Abstract In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl2 to the assay medium increased the ecto-ATPase activity in a dosedependent manner. At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46 mM MgCl2 . The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2 , but not by SrCl2 . The Mg2+ -dependent ATPase presents two apparent Km values for Mg-ATP2− (Km1 = 0.03 mM and Km2 = 2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg2+ -dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1 , ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60–70% of the Mg2+ -independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg2+ -dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4 -diisothiocyanostylbene-2 ,2 -disulfonic acid (DIDS) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+ -dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50 mM d-galactose. Since previous results showed that d-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg2+ -dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Tritrichomonas foetus; Ecto-ATPase; Ecto-phosphatase
∗ Corresponding author. Tel.: +55-21-590-4548; fax: +55-21-2270-8647. E-mail address: [email protected] (J.R. Meyer-Fernandes).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 5 7 6 - 3
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1. Introduction Trichomonads are flagellates which inhabit the alimentary canal or urogenital tract of vertebrates. The life cycles of the preponderant majority of trichomonads are simple, involving relatively monomorphic flagellate stages only, with four to six flagella. The trichomonad of cattle, Tritrichomonas foetus, and the human Trichomonas vaginalis are both parasite protozoans, exerting their pathogenic effect when interacting with the surface of epithelial cells. T. foetus is the etiologic agent of cattle sexually transmitted trichomoniosis, which is a severe veterinary disease, causing important economical losses (Honigberg, 1963). Surface membrane interactions between parasites and their host cells are of critical importance for the survival of the parasite, from both the immunological and physiological viewpoints (Alexander and Russel, 1992; Vannier-Santos et al., 1995; Martiny et al., 1996, 1999). Plasma membranes of cells contain enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells (Meyer-Fernandes et al., 1997; Furuya et al., 1998). Cell membrane ecto-ATPases are millimolar divalent cation-dependent, low specificity enzymes that hydrolyze all nucleotide triphosphates (Plesner, 1995; Zimmermann, 1999). The identity and the function of ecto-ATPases have been reviewed and the nomenclature of “E-type ATPases” was proposed to describe these enzymes (Plesner, 1995). Their physiological role is so far unknown. However, several hypothesis have been suggested, such as (i) protection from cytolytic effects of extracellular ATP (Filippini et al., 1990; Steinberg and Di Virgilio, 1991; Redegeld et al., 1991); (ii) regulation of ecto-kinase substrate concentration (Plesner, 1995); (iii) involvement in signal transduction (Margolis et al., 1990; Najjar et al., 1993; Dubyak and El-Moatassim, 1993; Clifford et al., 1997) and (iv) involvement in cellular adhesion (Knowles, 1995; Stout et al., 1995; Kirley, 1997). Ecto-ATPases have been described in some protozoa such as Toxoplasma gondii (Asai and Suzuki, 1990; Bermudes et al., 1994; Asai et al., 1995; Nakaar et al., 1998), Entamoeba histolytica (Barkker-Grunwald and Parduhn, 1993; Barros et al., 2000), Tetrahymena thermophila (Smith and Kirley, 1997), Leishmania tropica (Meyer-Fernandes et al., 1997), L. amazonensis (Berrêdo-Pinho et al., 2001) and Trypanosoma cruzi (Bernardes et al., 2000). A previous study on nucleotidases in the plasma membrane of T. foetus (Queiroz et al., 1991) reported the presence of a Mg2+ -dependent ATPase activity in the plasma membrane of this parasite. Here, we characterized the properties of an ecto-ATPase present on the surface of T. foetus: its divalent cation dependence, pH activation profile, specificity to nucleotides, sensitivity to suramin and modulation by d-galactose, a carbohydrate known to be exposed on the surface of host cells involved on T. foetus adhesion.
2. Materials and methods 2.1. Microorganism T. foetus, strain K (Queiroz et al., 1991), was kindly provided by Dr. Narcisa L. da Cunha e Silva, Instituto de Biof´ısica Carlos Chagas Filho, UFRJ, Brazil. The parasites were axenically maintained in TYM medium (Diamond, 1957), supplemented with 10% heat
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inactivated fetal bovine serum, for 24 h at 37 ◦ C. The cells at late logarithmic phase of growth were collected by centrifugation at 1400 × g for 5 min at 4 ◦ C and washed three times with 50 mM Hepes pH 7.0, 5.5 mM d-glucose, 5.4 mM KCl and 116 mM NaCl. Cellular viability was assessed, before and after incubations, by mobility and the Trypan blue method (Dutra et al., 1998). The viability of the cells was not affected under the conditions employed here. 2.2. Ecto-ATPase activity measurements Intact cells were incubated for 1 h at 36 ◦ C in 0.5 ml of a mixture containing, unless otherwise specified, 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose, 50.0 mM Hepes–Tris buffer, pH 7.2, 5.0 mM ATP, and 1.0 × 107 cells/ml, in the presence or in the absence of 5.0 mM MgCl2 . The ATPase activity was determined by measuring the hydrolysis of [␥-32 P]ATP (104 Bq/nmol ATP) (Saad-Nehme et al., 1997). The experiments were started by the addition of living cells and terminated by the addition of 1.0 ml of a cold mixture containing 0.2 g charcoal in 1.0 M HCl. The tubes were then centrifuged at 1500 × g for 10 min at 4 ◦ C. Aliquots (0.5 ml) of the supernatant containing the released 32 Pi were transferred to scintillation vials containing 9.0 ml of scintillation fluid (2.0 g PPO in 1.0 l of toluene). The ATPase activity was calculated by subtracting the nonspecific ATP hydrolysis measured in the absence of parasites. The ATP hydrolysis was linear with time under the assay conditions used and was proportional to the cell number. In the experiments where other nucleotides were used, the hydrolytic activity measured under the same conditions described above was assayed spectrophotometrically by measuring the release of Pi from the nucleotides (Lowry and Lopez, 1946). The hydrolysis of others nucleotides was also calculated by subtracting the nonspecific nucleotides hydrolysis measured in the absence of parasites. The values obtained for the ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Ca2+ , Mn2+ and Sr2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. 2.3. Phosphatase measurements In addition to the measurements of ecto-ATPase activity, the ecto-p-nitrophenylphosphatase activity was determined in the same medium as that for ATP hydrolysis except that ATP was replaced by 5.0 mM p-nitrophenylphosphate (p-NPP). The phosphatase activity was also calculated by subtracting the nonspecific p-NPP hydrolysis measured in the absence of parasites. The reaction was determined spectrophotometrically at 425.0 nm using an extinction coefficient of 14.3 × 103 M−1 cm−1 (Rodrigues et al., 1999). 2.4. Statistical analysis All experiments were performed in triplicate, with similar results obtained in at least three separate cell suspensions. Apparent Km and Vmax values were calculated using a
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computerized nonlinear regression analysis of the data to the Michaelis–Menten equation (Guilherme et al., 1991). Statistical significance was determined by Student’s t-test. Significance was considered as P < 0.05. 2.5. Reagents All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO). [␥-32 P]ATP was prepared as described by Glynn and Chappel (1964). Distilled water was deionized using a MilliQ system of resins (Millipore Corp., Bedford, MA) and was used in the preparation of all solutions. MgATP2− concentrations at equilibrium were calculated by using an iterative computer program that was modified (Sorenson et al., 1986) from that described by Fabiato and Fabiato (1979).
3. Results T. foetus presented a Mg2+ -dependent ecto-ATPase activity on its external surface. At pH 7.2, in the absence of any cation added (1 mM EDTA), T. foetus were able to hydrolyze ATP (44.0±3.9 nmol Pi/h, 107 cells), ADP (20.7±1.3 nmol Pi/h, 107 cells), AMP (9.9±1.1 nmol Pi/h, 107 cells) and p-NPP (10.6 ± 1.9 nmol Pi/h, 107 cells) (Fig. 1). The addition of 5 mM MgCl2 stimulated only the ATP hydrolysis (Fig. 1). The Mg2+ -dependent ecto-ATPase activity [difference between total (measured in the presence of 5 mM MgCl2 ) and basal
Fig. 1. Influence of MgCl2 on the ecto-phosphohydrolase activities of intact cells of T. foetus. Hachured bars: total activity, measured in the presence of 5 mM MgCl2 . Blank bars: basal activity, measured in the absence of MgCl2 . In these experiments, ATP hydrolysis was measured using the same colorimetric assay (described under Section 2) for Pi release as that used for other nucleotides. Data are means ± S.E. of three determinations with different cell suspensions.
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Fig. 2. Time course (A) and cell density dependence (B) of the Mg2+ -dependent ecto-ATPase activity of intact cells of T. foetus. Cells were incubated for different periods of time (A) or for 1 h (B) at 36 ◦ C. The Mg2+ -dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2 , minus the basal activity, measured in the absence of MgCl2 . Data are means ± S.E. of three determinations with different cell suspensions.
ecto-ATPase activity (measured in the absence of Mg Cl2 )] present in these parasites hydrolyzed ATP at 53.6 ± 6.3 nmol Pi/h, 107 cells. The time course of ATP hydrolysis by the Mg2+ -dependent ecto-ATPase present on the surface of T. foetus was linear for at least 60 min (Fig. 2A). Similarly, in assays to determine the influence of cell density, the Mg2+ -dependent activity measured over 60 min was linear over a nearly eight-fold range of cell density (Fig. 2B). The presence of a phosphatase activity on the external surface of T. foetus was confirmed by the ability of intact cells to hydrolyze p-NPP (Fig. 1). Therefore, to discard the possibility that the ATP hydrolysis stimulated by MgCl2 was due to phosphatase or other type of ATPases with internal ATP binding sites, different inhibitors for those enzymes were tested. Table 1 shows that the Mg2+ -dependent ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase (Meyer-Fernandes et al., 1997); bafilomycin A1 , a V-ATPase inhibitor (Browman et al., 1988); ouabain, a Na+ + K+ -ATPase inhibitor (Caruso-Neves et al., 1998a); furosemide, a Na+ -ATPase inhibitor (Caruso-Neves et al., 1998b) and vanadate, a potent inhibitor of P-ATPases and acid phosphatases (Fernandes et al., 1997; Dutra et al., 1998; Meyer-Fernandes et al., 1999). However, molybdate, which is a phosphatase inhibitor (Dutra et al., 1998), and vanadate inhibited the Mg2+ -independent ecto-ATPase, as well as the phosphatase activities (Table 1), suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. Levamizole, an inhibitor of alkaline phosphatase (Van Belle, 1976), and dipyridamole, a nucleoside transporter antagonist (Lemmens et al., 1996) failed to inhibit the ATPase and phosphatase activities (Table 1). To confirm that the ATP hydrolysis stimulated by MgCl2 was not due to other enzyme activities, the effect
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Table 1 Influence of various agents on ATPase and p-nitrophenylphosphatase activities of T. foetusa Additions
Control Levamizole (1.0 mM) Vanadate (1.0 mM) Molybdate (0.1 mM) Tartrate (1.0 mM) NaF (1.0 mM) Ouabain (1.0 mM) Azide (10.0 mM) Bafilomycin (1 nM) Oligomycin (1 g/ml) Furosemide (1.0 mM) Dipyridamole (10 M)
NPPase
100.0 ± 7.6 100.6 ± 6.3 30.6 ± 2.5 32.8 ± 2.2 84.4 ± 5.8 35.0 ± 2.5 102.6 ± 4.6 101.0 ± 3.6 100.5 ± 8.9 101.6 ± 3.8 102.0 ± 6.4 96.8 ± 9.6
ATPase Independent of MgCl2
Dependent on MgCl2
100.0 ± 5.9 100.8 ± 7.9 35.8 ± 8.5 39.5 ± 8.5 99.8 ± 8.1 79.1 ± 6.2 103.2 ± 9.7 96.9 ± 8.7 99.4 ± 6.0 98.7 ± 9.9 108.7 ± 7.5 101.2 ± 12.7
100.0 ± 4.3 100.1 ± 5.9 83.8 ± 6.6 82.7 ± 6.4 87.0 ± 9.6 89.4 ± 7.7 84.5 ± 6.9 95.8 ± 9.1 100.3 ± 8.3 91.3 ± 8.2 110.0 ± 6.1 116.9 ± 9.7
a Activities are expressed as a percentage of that measured under control conditions, i.e. without other additions. The Mg2+ -independent ATPase (47.4 ± 2.8 nmol Pi/h, 107 cells), the Mg2+ -dependent ATPase (58.2 ± 2.9 nmol Pi/h, 107 cells), and the p-NPPase (12.7 ± 0.96 nmol of Pi/h, 107 cells) activities were taken as 100%. The standard errors were calculated from the absolute activity values of three experiments, performed in triplicate, with different cell suspensions and converted to percentage of the control value. The unpaired t-test showed, in all cases with respect to Mg2+ -dependent ATPase activity, that there were not statistical differences (P > 0.05) with respect to values found for each compound.
of pH on the ecto-phosphohydrolase activities was analyzed in the pH range from 6.4 to 8.0, in which the cells were alive throughout the time course of reaction. The phosphatase, ADPase and 5 -nucleotidase activities decreased concomitantly with the increase of pH (Fig. 3A). Similarly, the ecto-ATPase independent of MgCl2 decreased with the increase of pH (Fig. 3B, closed circles). However, the Mg2+ -dependent ecto-ATPase in this pH range progressively increased to reach a maximal level at pH 8.0 (Fig. 3B, open circle). Mg2+ -dependent ecto-ATPase activities stimulated by an increase in pH have also been reported in L. tropica (Meyer-Fernandes et al., 1997) and in E. histolytica (Barros et al., 2000). Since we used intact cells for measuring the enzyme activities in all the experiments done in this work, it is likely that the described Mg2+ -dependent ATPase activity is an ecto-enzyme. To confirm this, we applied the criterion that an authentic ecto-enzyme should be inhibited by an added extracellular impermeant inhibitor (Knowles, 1988; Barbacci et al., 1996; Meyer-Fernandes et al., 1997; Barros et al., 2000), such as 4,4 -diisothiocyanostylbene-2 ,2 -disulfonic acid (DIDS) (Knowles, 1988; Barbacci et al., 1996; Meyer-Fernandes et al., 1997; Barros et al., 2000; Berrêdo-Pinho et al., 2001) and possibly by suramin, which is an ecto-ATPase inhibitor and also an antagonist of P2 -purinergic receptors (Hourani and Chown, 1989; Ziganshin et al., 1995). The Mg2+ -dependent ATPase activity was inhibited by DIDS in a dose-dependent manner. At 1 mM DIDS, the Mg2+ -dependent ATPase activity was inhibited by 63% and the Mg2+ -independent ATPase activity was inhibited by only 23% (Fig. 4A). We found that 1 mM suramin inhibited 87% of the Mg2+ -dependent ATPase activity and only 26% of the Mg2+ -independent ATPase activity (Fig. 4B).
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Fig. 3. Effect of pH on the ecto-phosphatase, ecto-ADPase and ecto-5 -nucleotidase activities (A) and on the ecto-ATPase activities (B) of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose, 1.0 × 107 cells/ml, 50 mM Hepes–Tris buffer, adjusted to pH values shown in the abscissa and 5 mM either p-NPP (䉲), ADP (䉱), or 5 -AMP (䊏) (A); or ATP (B), without (䊉) or with (䊊) the addition of 5 mM MgCl2 . In this pH range (6.4–8.0), the cells were viable throughout the course of the experiments. Data are means ± S.E. of three determinations with different cell suspensions.
Recently, it was shown that Mg2+ is an important extracellular signal in the regulation of Salmonella virulence (Véscovi et al., 1996). The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity of T. foetus in a dose-dependent manner (Fig. 5A). At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.47 mM MgCl2 . The addition of CaCl2 or MnCl2 to the extracellular medium also increased this ecto-ATPase in a dose-dependent manner (Fig. 5B and C, respectively), whereas high
Fig. 4. Effect of increasing concentrations of DIDS (A) and suramin (B) on the ecto-ATPase activities of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, without (䊉) or with (䊊) the addition of 5 mM MgCl2 with increasing concentrations of DIDS (A) or suramin (B). Data are means ± S.E. of three determinations with different cell suspensions.
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Fig. 5. Influence of different cation concentrations on the ecto-ATPase activities of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, with the addition of increasing concentrations of Mg2+ (A), Ca2+ (B), Mn2+ (C) or Sr2+ (D). Data are means±S.E. of three determinations with different cell suspensions.
concentrations of MnCl2 inhibited it. The stimulation observed with Mg2+ , Ca2+ and Mn2+ was not observed when these cations were replaced by Sr2+ (Fig. 5D). This Mg2+ -dependent ecto-ATPase revealed two kinetic components with respect to Mg-ATP2− concentrations (Fig. 6), one with a high affinity for Mg-ATP2− (K m = 0.03 mM) and one with low affinity for Mg-ATP2− (K m = 2.01 mM). We analyzed the specificity of these Mg2+ -dependent ecto-ATPase activity for other nucleotides. Table 2 shows that ATP was the best substrate for this enzyme and that this ecto-ATPase hydrolyzed ITP, CTP and UTP at high rates. GTP produced low reaction rate and ADP was not recognized as substrate for this enzyme, indicating that it is an ecto-ATPase (Heine et al., 1999) and not an ecto-ATP diphosphohydrolase, described for other cells (Handa and Guidotti, 1996; Wang and Guidotti, 1996). It is well known that carbohydrates exposed on the surface of mammalian cells play an important role in the process of interaction with T. foetus (Bonilha et al., 1995). Adhesins with lectin properties comparable with those reported for E. histolytica and Giardia lamblia
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Fig. 6. Dependence on Mg-ATP2− concentrations of the ecto-ATPase activity of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, which corresponds to Mg-ATP2− concentrations varying as shown on the abscissa. Curves represent the fit of experimental data by nonlinear regression using the Michaelis–Menten equation as described under Section 2. Inset: initial rates obtained with Mg-ATP2− concentrations varying between 0.01 and 0.5 mM (expanded scale). Data are means ± S.E. of three determinations with different cell suspensions.
have also been identified as having an important role in citoadherence of T. mobilensis to mammalian cells (Demes et al., 1989). The physiological role of the ecto-ATPases is unknown, but a possible involvement with cellular adhesion has been suggested (Knowles, 1995; Stout et al., 1995; Kirley, 1997). We have previously shown that d-galactose stimulated the Mg2+ -dependent ecto-ATPase activity present in E. histolytica (Barros et al., 2000). For these reasons, we examined the effect of some carbohydrates on the Mg2+ -dependent ecto-ATPase of T. foetus (Fig. 7). The ecto-ATPase was stimulated by more than 90% by 50 mM d-galactose and by 33% by 50 mM d-mannose (Fig. 7). d-Glucose had no effect on this hydrolytic activity (Fig. 7).
Table 2 Substrate specificity of Mg2+ -dependent ecto-ATPase activitya Nucleotides
Relative activity
ATP ITP CTP UTP GTP ADP
100.0 ± 2.2 80.9 ± 4.6 79.2 ± 2.0 76.9 ± 4.2 45.3 ± 5.1 0.0
a Reactions were performed at 36 ◦ C. The ATP hydrolysis (62.5 ± 3.4 nmol Pi/h, 107 cells) was taken as 100%. The standard errors were calculated from the absolute activity values of three experiments with different cell suspensions and converted to percentage of the control value.
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Fig. 7. Effects of carbohydrates on the ecto-ATPase activity of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, in the absence (control) or in the presence of 50 mM of the following carbohydrates: d-glucose (Glu), d-mannose (Man) and d-galactose (Gal). Data are means ± S.E. of three determinations with different cell suspensions.
4. Discussion In this paper, we report the characterization of a Mg2+ -dependent ecto-ATPase activity present on the external surface of T. foetus. Cellular integrity and viability were assessed, before and after the reactions, by the Trypan blue method (Dutra et al., 1998). The integrity of the cells was not affected by any conditions used in the assays. The external location of the ATP-hydrolyzing site is supported by its sensitivity to the impermeant inhibitor DIDS (Fig. 4A) (Knowles, 1988; Barbacci et al., 1996; Meyer-Fernandes et al., 1997; Barros et al., 2000), and to suramin (Fig. 4B), a noncompetitive inhibitor of some ecto-ATPases and an antagonist of P2 purinoreceptors, which mediate the physiological functions of extracellular ATP (Hourani and Chown, 1989; Ziganshin et al., 1995). To check the possibility that the observed ATP hydrolysis was the result of secreted soluble enzymes, as seen in other parasites (Bermudes et al., 1994; Smith and Kirley, 1997), we prepared a reaction mixture with cells that were incubated in the absence of ATP. Subsequently, the suspension was centrifuged to remove cells and the supernatant was checked for ATPase activity. This supernatant failed to show ATP hydrolysis either in the absence or in the presence of MgCl2 (data not shown). This data also rules out the possibility that the ATPase activity here described could be from lysed T. foetus cells. Also, the use of a battery of inhibitors for phosphatases and other ATPases that have intracellular ATP binding showed no effect on this ATPase activity (Table 1). For these reasons, we assign an ecto-localization of the Mg2+ -dependent ATPase activity described here (Plesner, 1995; Meyer-Fernandes et al., 1997).
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Here, we further characterize the T. foetus Mg2+ -dependent ecto-ATPase activity, showing that the addition of MgCl2 to the assay medium increased the ecto-ATPase activity in a dose-dependent manner (Fig. 5A). Ca2+ and Mn2+ also stimulated this enzyme (Fig. 5B and C), whereas Sr2+ was not able to replace these cations (Fig. 5D). Differences in the response to pH variation observed for the ATP hydrolysis in the absence or in the presence of Mg2+ (Fig. 3), as well as in their sensitivity to the inhibition by DIDS (Fig. 4A), suramin (Fig. 4B), vanadate and molybdate (Table 1) indicate that these activities are due to different enzymes. The optimum pH for the Mg2+ -dependent ecto-ATPase lies in the alkaline range (Fig. 3). Similar results were obtained for L. tropica (Meyer-Fernandes et al., 1997) and E. histolytica (Barros et al., 2000) Mg2+ -dependent ecto-ATPases, which also exhibit higher activity in alkaline pH and do not respond to phosphatase inhibitors (Meyer-Fernandes et al., 1997; Barros et al., 2000). It has been shown that the nucleoside triphosphate hydrolyse (NTPase) purified from T. gondii is not a single enzyme, but a mixture of two isozymes, termed NTPase I and NTPase II, and that a primary difference between these isozymes is that NTPase II hydrolyzes nucleoside triphosphate and diphosphate substrates at almost the same rate, whereas NTPase I was almost exclusively limited to nucleoside triphosphate hydrolysis (Asai et al., 1995). Recently, it has been shown that avirulent T. gondii strains express only NTPase II, whereas virulent strains express both NTPase I and NTPase II (Nakaar et al., 1998). The Mg2+ -dependent ecto-ATPase present in T. foetus hydrolyzes ATP, ITP, CTP and UTP at almost the same rate. It hydrolyzes GTP at a lower rate and ADP is not substrate for this enzyme. We have also shown that the Mg2+ -dependent ecto-ATPase present in L. tropica was much less active towards ADP than ATP (Meyer-Fernandes et al., 1997). Recently, we have shown that the invasive amoeba E. histolytica has much higher Mg2+ dependent ecto-ATP diphosphohydrolase activity than the noninvasive amoeba E. histolytica and the free-living amoeba E. moshkovskii (Barros et al., 2000). This amoeba E-type ATPase is stimulated by more than two-fold by d-galactose (Barros et al., 2000), an important molecule involved with E. histolytica adhesion (Radvin et al., 1980, 1989; Radvin and Guerrant, 1981), so we have proposed that this enzyme may be a pathogenesis marker for this cell (Barros et al., 2000). d-Galactose exposed on the surface of host cells is also involved on T. foetus adhesion (Bonilha et al., 1995). The Mg2+ -dependent ecto-ATPase of T. foetus here described and the Mg2+ -dependent ecto-ATP diphosphohydrolase of E. histolytica (Barros et al., 2000) share several characteristics, such as similar response to pH variation (Fig. 3B), sensitivity to the impermeant inhibitor DIDS and to suramin (Fig. 4A and B, respectively) and similar response to the stimulatory effect of d-galactose (Fig. 7). Taking these data into account, we suggest that these enzymes may have similar function in these parasites. As it has been suggested that E-type ATPases may be involved with cellular adhesion (Knowles, 1995; Stout et al., 1995; Kirley, 1997) it remains to be elucidated whether this Mg2+ -dependent ecto-ATPase may be associated with T. foetus infection.
Acknowledgements Dr. Narcisa L. da Cunha e Silva is gratefully acknowledged for providing us with the T. foetus strain used in this work. This work was partially supported by grants from the
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Brazilian Agencies, Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Programa de Núcleos de Excelência (PRONEX, grant 0885). References Alexander, J., Russel, D.G., 1992. The interaction of Leishmania species with macrophages. Adv. Parasitol. 31, 175–254. Asai, T., Suzuki, Y., 1990. Remarkable activities of nucleoside triphosphate hydrolase in tachyzoites of both virulent and avirulent strains of Toxoplasma gondii. FEMS Microbiol. Lett. 72, 89–92. Asai, T., Miura, S., Sibley, L.D., Okabayashi, H., Takeushi, T., 1995. Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii. J. Biol. Chem. 270, 1191–1197. Barbacci, E., Filippini, A., De Cesaris, P., Ziparo, E., 1996. Identification and characterization of an ecto-ATPase activity in rat sertoli cells. Biochem. Biophys. Res. Commun. 222, 273–279. Barkker-Grunwald, T., Parduhn, H., 1993. The Ca2+ -ATPase activity of Entamoeba histolytica is exposed towards the medium and towards the lumen of intracellular vesicles. Mol. Biochem. Parasitol. 57, 167–170. Barros, F.S., De Menezes, L.F., Pinheiro, A.A.S., Silva, E.F., Lopes, A.H.C.S., De Souza, W., Meyer-Fernandes, J.R., 2000. Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica. Arch. Biochem. Biophys. 375, 304–314. Bermudes, D., Peck, K.R., Afifi, M.A., Beckers, C.J.M., Joiner, K.A., 1994. Tandemly repeated genes encoded nucleoside triphosphate hydrolase isoforms secreted into parasitophorous vacuele of Toxoplasma gondii. J. Biol. Chem. 269, 2952–2960. Bernardes, C.F., Meyer-Fernandes, J.R., Saad-Nehme, J., Peres-Sampaio, C.E., Vannier-Santos, M.A., Vercesi, A.E., 2000. Int. J. Biochem. Cell. Biol. 32, 519–527. Berrêdo-Pinho, M., Peres-Sampaio, C.E., Chrispim, P.P.M., Belmont-Firpo, R., Lemos, A.P., Martiny, A., Vannier-Santos, M.A., Meyer-Fernandes, J.R., 2001. A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Arch. Biochem. Biophys. 391, 16–24. Bonilha, V.L., Clivaglia, M.C., De Souza, W., Silva Filho, F.C., 1995. The involvement of terminal carbohydrates of the mammalian cell surface in the cytoadhesion of Trichomonads. Parasitol. Res. 81, 121–126. Browman, E.J., Siebers, A., Altendorf, K., 1988. Bafilomycin: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. U.S.A. 85, 7972–7976. Caruso-Neves, C., Meyer-Fernandes, J.R., Saad-Nehme, J., Lopes, A.G., 1998a. Osmotic modulation of the ouabain-sensitive (Na+ + K+ ) ATPase from malpighian tubules of Rhodnius prolixus. Z. Naturforsch. 53c, 911–917. Caruso-Neves, C., Meyer-Fernandes, J.R., Saad-Nehme, J., Proverbio, F., Mar´ın, R., Lopes, A.G., 1998b. Ouabain-insensitive Na+ -ATPase activity of malpighian tubules from Rhodnius prolixux. Comp. Biochem. Physiol. B 119, 807–811. Clifford, E.E., Martin, K.A., Dalal, P., Thomas, R., Dubyak, G.R., 1997. Stage-specific expression of P2Y receptors, ecto-apyrase and 5 -nucleotidase in myeloid leukocytes. Am. J. Physiol. 42, C973–C987. Demes, P., Pindak, F.F., Wells, D.J., Gardner Jr., W.A., 1989. Adherence surface properties of Tritrichomonas mobilensis, an intestinal parasite of the squirrel monkey. Parasitol. Res. 75, 589–594. Diamond, L.S., 1957. The establishment of various trichomonads of animals and men in axenic culture. J. Parasitol. 43, 488–490. Dubyak, G.R., El-Moatassim, C., 1993. Signal transduction via P2 -purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265, C577–C606. Dutra, P.M.L., Rodrigues, C.O., Jesus, J.B., Lopes, A.H.C.S., Souto-Padrón, T., Meyer-Fernandes, J.R., 1998. A novel ecto-phosphatase activity of Herpetomonas muscarum muscarum inhibited by platelet-activating factor. Biochem. Biophys. Res. Commun. 253, 164–169. Fabiato, A., Fabiato, F., 1979. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. (Paris) 75, 463–505.
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Fernandes, E.C., Meyer-Fernandes, J.R., Silva-Neto, M.A.C., Vercesi, A.E., 1997. Trypanosoma brucei: ecto-phosphatase activity on the surface of intact procyclic forms. Z. Naturforsch. 52c, 351–358. Filippini, A., Taffs, R.E., Agui, T., Sitkovsky, M.V., 1990. Ecto-ATPase activity in cytolytic T-lymphocytes. J. Biol. Chem. 265, 334–340. Furuya, T., Zhong, L., Meyer-Fernandes, J.R., Lu, H.-G., Moreno, S.N.J., Docampo, R., 1998. Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective forms. Mol. Biochem. Parasitol. 92, 339–348. Glynn, I.M., Chappel, J.B., 1964. A simple method for the preparation of 32 P-labelled adenosine triphosphate of high specific activity. Biochem. J. 90, 147–149. Guilherme, A., Meyer-Fernandes, J.R., Vieyra, A., 1991. Reversible inhibition by 4,4 -diisothiocyanatostilbene2,2 -disulfonic acid of the plasma membrane (Ca2+ + Mg2+ ) ATPase from kidney proximal tubules. Biochemistry 30, 5700–5706. Handa, M., Guidotti, G., 1996. Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potate tubers (Solanum tuberosum). Biochem. Biophys. Res. Commun. 218, 916–923. Heine, P., Braun, N., Heilbronn, A., Zimmermann, H., 1999. Functional characterization of rat ecto-ATPase and ecto-ATP diphosphohydrolase after heterologous expression in CHO cells. Eur. J. Biochem. 262, 102–107. Honigberg, B.M., 1963. Evolutionary and systematic relationships in flagellate order Trichomonadida Kirby. J. Protozool. 10, 20–63. Hourani, S.M.O., Chown, J.A., 1989. The effects of some possible inhibitors of ecto-nucleotidases on the breakdown and pharmacology effects of ATP in the guinea-pig urinary bladder. Gen. Pharmacol. 20, 413–416. Kirley, T.L., 1997. Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase. J. Biol. Chem. 272, 1076–1081. Knowles, A.F., 1988. Differential expression of ecto-Mg 2+ -ATPase and ecto-Ca2+ -ATPase activities in human hepatoma cells. Arch. Biochem. Biophys. 263, 264–271. Knowles, A.F., 1995. The rat liver ecto-ATPase/C-CAM cDNA detects induction of carcinoembryonic antigen but not the mercurial-insensitive ecto-ATPase in human hepatoma Li-7A cells treated by epidermal growth factor and cholera toxin. Biochem. Biophys. Res. Commun. 207, 529–535. Lemmens, R., Vanduffell, L., Teuchy, H., Culic, O., 1996. Regulation of proliferation of LLC-MK2 cells by nucleosides and nucleotides: the role of ecto-enzymes. Biochem. J. 316, 551–557. Lowry, H.O., Lopez, M., 1946. The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem. 162, 421–428. Margolis, R.N., Schell, M.J., Taylor, S.L., Hubbard, A.L., 1990. Hepatocyte plasma membrane ecto-ATPase (pp120/HA4) is a substrate for tyrosine kinase activity of the insulin receptor. Biochem. Biophys. Res. Commun. 166, 562–566. Martiny, A., Vannier-Santos, M.A., Borges, V.M., Meyer-Fernandes, J.R., Asrreuy, J., Cunha e Silva, N.L., De Souza, W., 1996. Leishmania induced tyrosine phosphorylation in the host macrophage and its implication to infection. Eur. J. Cell Biol. 71, 206–215. Martiny, A., Meyer-Fernandes, J.R., De Souza, W., Vannier-Santos, M.A., 1999. Altered tyrosine phosphorylation of ERK1 MAP kinase and other macrophage molecules caused by Leishmania amastigotes. Mol. Biochem. Parasitol. 102, 1–12. Meyer-Fernandes, J.R., Vieyra, A., 1988. Pyrophosphate formation from acetyl phosphate and ortophosphate: evidence for heterogeneous catalysis. Arch. Biochem. Biophys. 266, 132–141. Meyer-Fernandes, J.R., Dutra, P.M.L., Rodrigues, C.O., Saad-Nehme, J., Lopes, A.H.C.S., 1997. Mg-dependent ecto-ATPase activity in Leishmania tropica. Arch. Biochem. Biophys. 341, 40–46. Meyer-Fernandes, J.R., Silva-Neto, M.A.C., Soares, M.S., Fernandes, E., Vercesi, A.E., Oliveira, M.M., 1999. Ecto-phosphatase activities on the cell surface of the amastigotes forms of Trypanosoma cruzi. Z. Naturforsch. 54c, 977–984. Najjar, S.M., Accili, D., Philippe, N., Jernberg, J., Margolis, R., Taylor, S.I., 1993. PP 120/ecto-ATPase, an endogenous substrate of the insulin receptor tyrosine kinase, is expressed as two variably spliced isoforms. J. Biol. Chem. 268, 1201–1206. Nakaar, V., Beckers, C.J.M., Polotsky, V., Joiner, K.A., 1998. Basis for substrate specificity of the Toxoplasma gondii nucleotide triphosphate hydrolase. Mol. Biochem. Parasitol. 97, 209–220. Plesner, L., 1995. Ecto-ATPases: identities and functions. Int. Rev. Cytol. 158, 141–214. Queiroz, R.C.B., Silva Santos, L.M., Benchimol, M., De Souza, W., 1991. Cytochemical localization of enzyme markers in Tritrichomonas foetus. Parasitol. Res. 77, 561–566.
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Characterization of an ecto-ATPase of Tritrichomonas foetus José B. Jesus a,b , Angela H.C.S. Lopes b , José R. Meyer-Fernandes a,∗ a
Departamento de Bioqu´ımica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21541-590 Rio de Janeiro, RJ, Brazil b Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio de Janeiro, RJ, Brazil Received 1 June 2001; received in revised form 29 August 2001; accepted 29 August 2001
Abstract In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl2 to the assay medium increased the ecto-ATPase activity in a dosedependent manner. At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46 mM MgCl2 . The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2 , but not by SrCl2 . The Mg2+ -dependent ATPase presents two apparent Km values for Mg-ATP2− (Km1 = 0.03 mM and Km2 = 2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg2+ -dependent ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1 , ouabain, furosemide, vanadate, molybdate, sodium fluoride and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60–70% of the Mg2+ -independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg2+ -dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4 -diisothiocyanostylbene-2 ,2 -disulfonic acid (DIDS) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+ -dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50 mM d-galactose. Since previous results showed that d-galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg2+ -dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Tritrichomonas foetus; Ecto-ATPase; Ecto-phosphatase
∗ Corresponding author. Tel.: +55-21-590-4548; fax: +55-21-2270-8647. E-mail address: [email protected] (J.R. Meyer-Fernandes).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 1 ) 0 0 5 7 6 - 3
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1. Introduction Trichomonads are flagellates which inhabit the alimentary canal or urogenital tract of vertebrates. The life cycles of the preponderant majority of trichomonads are simple, involving relatively monomorphic flagellate stages only, with four to six flagella. The trichomonad of cattle, Tritrichomonas foetus, and the human Trichomonas vaginalis are both parasite protozoans, exerting their pathogenic effect when interacting with the surface of epithelial cells. T. foetus is the etiologic agent of cattle sexually transmitted trichomoniosis, which is a severe veterinary disease, causing important economical losses (Honigberg, 1963). Surface membrane interactions between parasites and their host cells are of critical importance for the survival of the parasite, from both the immunological and physiological viewpoints (Alexander and Russel, 1992; Vannier-Santos et al., 1995; Martiny et al., 1996, 1999). Plasma membranes of cells contain enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ecto-enzymes, can be measured using living cells (Meyer-Fernandes et al., 1997; Furuya et al., 1998). Cell membrane ecto-ATPases are millimolar divalent cation-dependent, low specificity enzymes that hydrolyze all nucleotide triphosphates (Plesner, 1995; Zimmermann, 1999). The identity and the function of ecto-ATPases have been reviewed and the nomenclature of “E-type ATPases” was proposed to describe these enzymes (Plesner, 1995). Their physiological role is so far unknown. However, several hypothesis have been suggested, such as (i) protection from cytolytic effects of extracellular ATP (Filippini et al., 1990; Steinberg and Di Virgilio, 1991; Redegeld et al., 1991); (ii) regulation of ecto-kinase substrate concentration (Plesner, 1995); (iii) involvement in signal transduction (Margolis et al., 1990; Najjar et al., 1993; Dubyak and El-Moatassim, 1993; Clifford et al., 1997) and (iv) involvement in cellular adhesion (Knowles, 1995; Stout et al., 1995; Kirley, 1997). Ecto-ATPases have been described in some protozoa such as Toxoplasma gondii (Asai and Suzuki, 1990; Bermudes et al., 1994; Asai et al., 1995; Nakaar et al., 1998), Entamoeba histolytica (Barkker-Grunwald and Parduhn, 1993; Barros et al., 2000), Tetrahymena thermophila (Smith and Kirley, 1997), Leishmania tropica (Meyer-Fernandes et al., 1997), L. amazonensis (Berrêdo-Pinho et al., 2001) and Trypanosoma cruzi (Bernardes et al., 2000). A previous study on nucleotidases in the plasma membrane of T. foetus (Queiroz et al., 1991) reported the presence of a Mg2+ -dependent ATPase activity in the plasma membrane of this parasite. Here, we characterized the properties of an ecto-ATPase present on the surface of T. foetus: its divalent cation dependence, pH activation profile, specificity to nucleotides, sensitivity to suramin and modulation by d-galactose, a carbohydrate known to be exposed on the surface of host cells involved on T. foetus adhesion.
2. Materials and methods 2.1. Microorganism T. foetus, strain K (Queiroz et al., 1991), was kindly provided by Dr. Narcisa L. da Cunha e Silva, Instituto de Biof´ısica Carlos Chagas Filho, UFRJ, Brazil. The parasites were axenically maintained in TYM medium (Diamond, 1957), supplemented with 10% heat
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inactivated fetal bovine serum, for 24 h at 37 ◦ C. The cells at late logarithmic phase of growth were collected by centrifugation at 1400 × g for 5 min at 4 ◦ C and washed three times with 50 mM Hepes pH 7.0, 5.5 mM d-glucose, 5.4 mM KCl and 116 mM NaCl. Cellular viability was assessed, before and after incubations, by mobility and the Trypan blue method (Dutra et al., 1998). The viability of the cells was not affected under the conditions employed here. 2.2. Ecto-ATPase activity measurements Intact cells were incubated for 1 h at 36 ◦ C in 0.5 ml of a mixture containing, unless otherwise specified, 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose, 50.0 mM Hepes–Tris buffer, pH 7.2, 5.0 mM ATP, and 1.0 × 107 cells/ml, in the presence or in the absence of 5.0 mM MgCl2 . The ATPase activity was determined by measuring the hydrolysis of [␥-32 P]ATP (104 Bq/nmol ATP) (Saad-Nehme et al., 1997). The experiments were started by the addition of living cells and terminated by the addition of 1.0 ml of a cold mixture containing 0.2 g charcoal in 1.0 M HCl. The tubes were then centrifuged at 1500 × g for 10 min at 4 ◦ C. Aliquots (0.5 ml) of the supernatant containing the released 32 Pi were transferred to scintillation vials containing 9.0 ml of scintillation fluid (2.0 g PPO in 1.0 l of toluene). The ATPase activity was calculated by subtracting the nonspecific ATP hydrolysis measured in the absence of parasites. The ATP hydrolysis was linear with time under the assay conditions used and was proportional to the cell number. In the experiments where other nucleotides were used, the hydrolytic activity measured under the same conditions described above was assayed spectrophotometrically by measuring the release of Pi from the nucleotides (Lowry and Lopez, 1946). The hydrolysis of others nucleotides was also calculated by subtracting the nonspecific nucleotides hydrolysis measured in the absence of parasites. The values obtained for the ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same. In the experiments where high concentrations of Ca2+ , Mn2+ and Sr2+ were tested, possible precipitates formed were checked as previously described (Meyer-Fernandes and Vieyra, 1988). Under the conditions employed, in the reaction medium containing 50 mM Hepes pH 7.2, 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose and 5 mM ATP, no phosphate precipitates were observed in the presence of these cations. 2.3. Phosphatase measurements In addition to the measurements of ecto-ATPase activity, the ecto-p-nitrophenylphosphatase activity was determined in the same medium as that for ATP hydrolysis except that ATP was replaced by 5.0 mM p-nitrophenylphosphate (p-NPP). The phosphatase activity was also calculated by subtracting the nonspecific p-NPP hydrolysis measured in the absence of parasites. The reaction was determined spectrophotometrically at 425.0 nm using an extinction coefficient of 14.3 × 103 M−1 cm−1 (Rodrigues et al., 1999). 2.4. Statistical analysis All experiments were performed in triplicate, with similar results obtained in at least three separate cell suspensions. Apparent Km and Vmax values were calculated using a
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computerized nonlinear regression analysis of the data to the Michaelis–Menten equation (Guilherme et al., 1991). Statistical significance was determined by Student’s t-test. Significance was considered as P < 0.05. 2.5. Reagents All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO). [␥-32 P]ATP was prepared as described by Glynn and Chappel (1964). Distilled water was deionized using a MilliQ system of resins (Millipore Corp., Bedford, MA) and was used in the preparation of all solutions. MgATP2− concentrations at equilibrium were calculated by using an iterative computer program that was modified (Sorenson et al., 1986) from that described by Fabiato and Fabiato (1979).
3. Results T. foetus presented a Mg2+ -dependent ecto-ATPase activity on its external surface. At pH 7.2, in the absence of any cation added (1 mM EDTA), T. foetus were able to hydrolyze ATP (44.0±3.9 nmol Pi/h, 107 cells), ADP (20.7±1.3 nmol Pi/h, 107 cells), AMP (9.9±1.1 nmol Pi/h, 107 cells) and p-NPP (10.6 ± 1.9 nmol Pi/h, 107 cells) (Fig. 1). The addition of 5 mM MgCl2 stimulated only the ATP hydrolysis (Fig. 1). The Mg2+ -dependent ecto-ATPase activity [difference between total (measured in the presence of 5 mM MgCl2 ) and basal
Fig. 1. Influence of MgCl2 on the ecto-phosphohydrolase activities of intact cells of T. foetus. Hachured bars: total activity, measured in the presence of 5 mM MgCl2 . Blank bars: basal activity, measured in the absence of MgCl2 . In these experiments, ATP hydrolysis was measured using the same colorimetric assay (described under Section 2) for Pi release as that used for other nucleotides. Data are means ± S.E. of three determinations with different cell suspensions.
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Fig. 2. Time course (A) and cell density dependence (B) of the Mg2+ -dependent ecto-ATPase activity of intact cells of T. foetus. Cells were incubated for different periods of time (A) or for 1 h (B) at 36 ◦ C. The Mg2+ -dependent ecto-ATPase activity was calculated from the total activity, measured in the presence of 5 mM MgCl2 , minus the basal activity, measured in the absence of MgCl2 . Data are means ± S.E. of three determinations with different cell suspensions.
ecto-ATPase activity (measured in the absence of Mg Cl2 )] present in these parasites hydrolyzed ATP at 53.6 ± 6.3 nmol Pi/h, 107 cells. The time course of ATP hydrolysis by the Mg2+ -dependent ecto-ATPase present on the surface of T. foetus was linear for at least 60 min (Fig. 2A). Similarly, in assays to determine the influence of cell density, the Mg2+ -dependent activity measured over 60 min was linear over a nearly eight-fold range of cell density (Fig. 2B). The presence of a phosphatase activity on the external surface of T. foetus was confirmed by the ability of intact cells to hydrolyze p-NPP (Fig. 1). Therefore, to discard the possibility that the ATP hydrolysis stimulated by MgCl2 was due to phosphatase or other type of ATPases with internal ATP binding sites, different inhibitors for those enzymes were tested. Table 1 shows that the Mg2+ -dependent ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase (Meyer-Fernandes et al., 1997); bafilomycin A1 , a V-ATPase inhibitor (Browman et al., 1988); ouabain, a Na+ + K+ -ATPase inhibitor (Caruso-Neves et al., 1998a); furosemide, a Na+ -ATPase inhibitor (Caruso-Neves et al., 1998b) and vanadate, a potent inhibitor of P-ATPases and acid phosphatases (Fernandes et al., 1997; Dutra et al., 1998; Meyer-Fernandes et al., 1999). However, molybdate, which is a phosphatase inhibitor (Dutra et al., 1998), and vanadate inhibited the Mg2+ -independent ecto-ATPase, as well as the phosphatase activities (Table 1), suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. Levamizole, an inhibitor of alkaline phosphatase (Van Belle, 1976), and dipyridamole, a nucleoside transporter antagonist (Lemmens et al., 1996) failed to inhibit the ATPase and phosphatase activities (Table 1). To confirm that the ATP hydrolysis stimulated by MgCl2 was not due to other enzyme activities, the effect
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Table 1 Influence of various agents on ATPase and p-nitrophenylphosphatase activities of T. foetusa Additions
Control Levamizole (1.0 mM) Vanadate (1.0 mM) Molybdate (0.1 mM) Tartrate (1.0 mM) NaF (1.0 mM) Ouabain (1.0 mM) Azide (10.0 mM) Bafilomycin (1 nM) Oligomycin (1 g/ml) Furosemide (1.0 mM) Dipyridamole (10 M)
NPPase
100.0 ± 7.6 100.6 ± 6.3 30.6 ± 2.5 32.8 ± 2.2 84.4 ± 5.8 35.0 ± 2.5 102.6 ± 4.6 101.0 ± 3.6 100.5 ± 8.9 101.6 ± 3.8 102.0 ± 6.4 96.8 ± 9.6
ATPase Independent of MgCl2
Dependent on MgCl2
100.0 ± 5.9 100.8 ± 7.9 35.8 ± 8.5 39.5 ± 8.5 99.8 ± 8.1 79.1 ± 6.2 103.2 ± 9.7 96.9 ± 8.7 99.4 ± 6.0 98.7 ± 9.9 108.7 ± 7.5 101.2 ± 12.7
100.0 ± 4.3 100.1 ± 5.9 83.8 ± 6.6 82.7 ± 6.4 87.0 ± 9.6 89.4 ± 7.7 84.5 ± 6.9 95.8 ± 9.1 100.3 ± 8.3 91.3 ± 8.2 110.0 ± 6.1 116.9 ± 9.7
a Activities are expressed as a percentage of that measured under control conditions, i.e. without other additions. The Mg2+ -independent ATPase (47.4 ± 2.8 nmol Pi/h, 107 cells), the Mg2+ -dependent ATPase (58.2 ± 2.9 nmol Pi/h, 107 cells), and the p-NPPase (12.7 ± 0.96 nmol of Pi/h, 107 cells) activities were taken as 100%. The standard errors were calculated from the absolute activity values of three experiments, performed in triplicate, with different cell suspensions and converted to percentage of the control value. The unpaired t-test showed, in all cases with respect to Mg2+ -dependent ATPase activity, that there were not statistical differences (P > 0.05) with respect to values found for each compound.
of pH on the ecto-phosphohydrolase activities was analyzed in the pH range from 6.4 to 8.0, in which the cells were alive throughout the time course of reaction. The phosphatase, ADPase and 5 -nucleotidase activities decreased concomitantly with the increase of pH (Fig. 3A). Similarly, the ecto-ATPase independent of MgCl2 decreased with the increase of pH (Fig. 3B, closed circles). However, the Mg2+ -dependent ecto-ATPase in this pH range progressively increased to reach a maximal level at pH 8.0 (Fig. 3B, open circle). Mg2+ -dependent ecto-ATPase activities stimulated by an increase in pH have also been reported in L. tropica (Meyer-Fernandes et al., 1997) and in E. histolytica (Barros et al., 2000). Since we used intact cells for measuring the enzyme activities in all the experiments done in this work, it is likely that the described Mg2+ -dependent ATPase activity is an ecto-enzyme. To confirm this, we applied the criterion that an authentic ecto-enzyme should be inhibited by an added extracellular impermeant inhibitor (Knowles, 1988; Barbacci et al., 1996; Meyer-Fernandes et al., 1997; Barros et al., 2000), such as 4,4 -diisothiocyanostylbene-2 ,2 -disulfonic acid (DIDS) (Knowles, 1988; Barbacci et al., 1996; Meyer-Fernandes et al., 1997; Barros et al., 2000; Berrêdo-Pinho et al., 2001) and possibly by suramin, which is an ecto-ATPase inhibitor and also an antagonist of P2 -purinergic receptors (Hourani and Chown, 1989; Ziganshin et al., 1995). The Mg2+ -dependent ATPase activity was inhibited by DIDS in a dose-dependent manner. At 1 mM DIDS, the Mg2+ -dependent ATPase activity was inhibited by 63% and the Mg2+ -independent ATPase activity was inhibited by only 23% (Fig. 4A). We found that 1 mM suramin inhibited 87% of the Mg2+ -dependent ATPase activity and only 26% of the Mg2+ -independent ATPase activity (Fig. 4B).
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Fig. 3. Effect of pH on the ecto-phosphatase, ecto-ADPase and ecto-5 -nucleotidase activities (A) and on the ecto-ATPase activities (B) of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM d-glucose, 1.0 × 107 cells/ml, 50 mM Hepes–Tris buffer, adjusted to pH values shown in the abscissa and 5 mM either p-NPP (䉲), ADP (䉱), or 5 -AMP (䊏) (A); or ATP (B), without (䊉) or with (䊊) the addition of 5 mM MgCl2 . In this pH range (6.4–8.0), the cells were viable throughout the course of the experiments. Data are means ± S.E. of three determinations with different cell suspensions.
Recently, it was shown that Mg2+ is an important extracellular signal in the regulation of Salmonella virulence (Véscovi et al., 1996). The addition of MgCl2 to the extracellular medium increased the ecto-ATPase activity of T. foetus in a dose-dependent manner (Fig. 5A). At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.47 mM MgCl2 . The addition of CaCl2 or MnCl2 to the extracellular medium also increased this ecto-ATPase in a dose-dependent manner (Fig. 5B and C, respectively), whereas high
Fig. 4. Effect of increasing concentrations of DIDS (A) and suramin (B) on the ecto-ATPase activities of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, without (䊉) or with (䊊) the addition of 5 mM MgCl2 with increasing concentrations of DIDS (A) or suramin (B). Data are means ± S.E. of three determinations with different cell suspensions.
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Fig. 5. Influence of different cation concentrations on the ecto-ATPase activities of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, with the addition of increasing concentrations of Mg2+ (A), Ca2+ (B), Mn2+ (C) or Sr2+ (D). Data are means±S.E. of three determinations with different cell suspensions.
concentrations of MnCl2 inhibited it. The stimulation observed with Mg2+ , Ca2+ and Mn2+ was not observed when these cations were replaced by Sr2+ (Fig. 5D). This Mg2+ -dependent ecto-ATPase revealed two kinetic components with respect to Mg-ATP2− concentrations (Fig. 6), one with a high affinity for Mg-ATP2− (K m = 0.03 mM) and one with low affinity for Mg-ATP2− (K m = 2.01 mM). We analyzed the specificity of these Mg2+ -dependent ecto-ATPase activity for other nucleotides. Table 2 shows that ATP was the best substrate for this enzyme and that this ecto-ATPase hydrolyzed ITP, CTP and UTP at high rates. GTP produced low reaction rate and ADP was not recognized as substrate for this enzyme, indicating that it is an ecto-ATPase (Heine et al., 1999) and not an ecto-ATP diphosphohydrolase, described for other cells (Handa and Guidotti, 1996; Wang and Guidotti, 1996). It is well known that carbohydrates exposed on the surface of mammalian cells play an important role in the process of interaction with T. foetus (Bonilha et al., 1995). Adhesins with lectin properties comparable with those reported for E. histolytica and Giardia lamblia
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Fig. 6. Dependence on Mg-ATP2− concentrations of the ecto-ATPase activity of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, which corresponds to Mg-ATP2− concentrations varying as shown on the abscissa. Curves represent the fit of experimental data by nonlinear regression using the Michaelis–Menten equation as described under Section 2. Inset: initial rates obtained with Mg-ATP2− concentrations varying between 0.01 and 0.5 mM (expanded scale). Data are means ± S.E. of three determinations with different cell suspensions.
have also been identified as having an important role in citoadherence of T. mobilensis to mammalian cells (Demes et al., 1989). The physiological role of the ecto-ATPases is unknown, but a possible involvement with cellular adhesion has been suggested (Knowles, 1995; Stout et al., 1995; Kirley, 1997). We have previously shown that d-galactose stimulated the Mg2+ -dependent ecto-ATPase activity present in E. histolytica (Barros et al., 2000). For these reasons, we examined the effect of some carbohydrates on the Mg2+ -dependent ecto-ATPase of T. foetus (Fig. 7). The ecto-ATPase was stimulated by more than 90% by 50 mM d-galactose and by 33% by 50 mM d-mannose (Fig. 7). d-Glucose had no effect on this hydrolytic activity (Fig. 7).
Table 2 Substrate specificity of Mg2+ -dependent ecto-ATPase activitya Nucleotides
Relative activity
ATP ITP CTP UTP GTP ADP
100.0 ± 2.2 80.9 ± 4.6 79.2 ± 2.0 76.9 ± 4.2 45.3 ± 5.1 0.0
a Reactions were performed at 36 ◦ C. The ATP hydrolysis (62.5 ± 3.4 nmol Pi/h, 107 cells) was taken as 100%. The standard errors were calculated from the absolute activity values of three experiments with different cell suspensions and converted to percentage of the control value.
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Fig. 7. Effects of carbohydrates on the ecto-ATPase activity of intact cells of T. foetus. Cells were incubated for 1 h at 36 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in Section 2, in the absence (control) or in the presence of 50 mM of the following carbohydrates: d-glucose (Glu), d-mannose (Man) and d-galactose (Gal). Data are means ± S.E. of three determinations with different cell suspensions.
4. Discussion In this paper, we report the characterization of a Mg2+ -dependent ecto-ATPase activity present on the external surface of T. foetus. Cellular integrity and viability were assessed, before and after the reactions, by the Trypan blue method (Dutra et al., 1998). The integrity of the cells was not affected by any conditions used in the assays. The external location of the ATP-hydrolyzing site is supported by its sensitivity to the impermeant inhibitor DIDS (Fig. 4A) (Knowles, 1988; Barbacci et al., 1996; Meyer-Fernandes et al., 1997; Barros et al., 2000), and to suramin (Fig. 4B), a noncompetitive inhibitor of some ecto-ATPases and an antagonist of P2 purinoreceptors, which mediate the physiological functions of extracellular ATP (Hourani and Chown, 1989; Ziganshin et al., 1995). To check the possibility that the observed ATP hydrolysis was the result of secreted soluble enzymes, as seen in other parasites (Bermudes et al., 1994; Smith and Kirley, 1997), we prepared a reaction mixture with cells that were incubated in the absence of ATP. Subsequently, the suspension was centrifuged to remove cells and the supernatant was checked for ATPase activity. This supernatant failed to show ATP hydrolysis either in the absence or in the presence of MgCl2 (data not shown). This data also rules out the possibility that the ATPase activity here described could be from lysed T. foetus cells. Also, the use of a battery of inhibitors for phosphatases and other ATPases that have intracellular ATP binding showed no effect on this ATPase activity (Table 1). For these reasons, we assign an ecto-localization of the Mg2+ -dependent ATPase activity described here (Plesner, 1995; Meyer-Fernandes et al., 1997).
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Here, we further characterize the T. foetus Mg2+ -dependent ecto-ATPase activity, showing that the addition of MgCl2 to the assay medium increased the ecto-ATPase activity in a dose-dependent manner (Fig. 5A). Ca2+ and Mn2+ also stimulated this enzyme (Fig. 5B and C), whereas Sr2+ was not able to replace these cations (Fig. 5D). Differences in the response to pH variation observed for the ATP hydrolysis in the absence or in the presence of Mg2+ (Fig. 3), as well as in their sensitivity to the inhibition by DIDS (Fig. 4A), suramin (Fig. 4B), vanadate and molybdate (Table 1) indicate that these activities are due to different enzymes. The optimum pH for the Mg2+ -dependent ecto-ATPase lies in the alkaline range (Fig. 3). Similar results were obtained for L. tropica (Meyer-Fernandes et al., 1997) and E. histolytica (Barros et al., 2000) Mg2+ -dependent ecto-ATPases, which also exhibit higher activity in alkaline pH and do not respond to phosphatase inhibitors (Meyer-Fernandes et al., 1997; Barros et al., 2000). It has been shown that the nucleoside triphosphate hydrolyse (NTPase) purified from T. gondii is not a single enzyme, but a mixture of two isozymes, termed NTPase I and NTPase II, and that a primary difference between these isozymes is that NTPase II hydrolyzes nucleoside triphosphate and diphosphate substrates at almost the same rate, whereas NTPase I was almost exclusively limited to nucleoside triphosphate hydrolysis (Asai et al., 1995). Recently, it has been shown that avirulent T. gondii strains express only NTPase II, whereas virulent strains express both NTPase I and NTPase II (Nakaar et al., 1998). The Mg2+ -dependent ecto-ATPase present in T. foetus hydrolyzes ATP, ITP, CTP and UTP at almost the same rate. It hydrolyzes GTP at a lower rate and ADP is not substrate for this enzyme. We have also shown that the Mg2+ -dependent ecto-ATPase present in L. tropica was much less active towards ADP than ATP (Meyer-Fernandes et al., 1997). Recently, we have shown that the invasive amoeba E. histolytica has much higher Mg2+ dependent ecto-ATP diphosphohydrolase activity than the noninvasive amoeba E. histolytica and the free-living amoeba E. moshkovskii (Barros et al., 2000). This amoeba E-type ATPase is stimulated by more than two-fold by d-galactose (Barros et al., 2000), an important molecule involved with E. histolytica adhesion (Radvin et al., 1980, 1989; Radvin and Guerrant, 1981), so we have proposed that this enzyme may be a pathogenesis marker for this cell (Barros et al., 2000). d-Galactose exposed on the surface of host cells is also involved on T. foetus adhesion (Bonilha et al., 1995). The Mg2+ -dependent ecto-ATPase of T. foetus here described and the Mg2+ -dependent ecto-ATP diphosphohydrolase of E. histolytica (Barros et al., 2000) share several characteristics, such as similar response to pH variation (Fig. 3B), sensitivity to the impermeant inhibitor DIDS and to suramin (Fig. 4A and B, respectively) and similar response to the stimulatory effect of d-galactose (Fig. 7). Taking these data into account, we suggest that these enzymes may have similar function in these parasites. As it has been suggested that E-type ATPases may be involved with cellular adhesion (Knowles, 1995; Stout et al., 1995; Kirley, 1997) it remains to be elucidated whether this Mg2+ -dependent ecto-ATPase may be associated with T. foetus infection.
Acknowledgements Dr. Narcisa L. da Cunha e Silva is gratefully acknowledged for providing us with the T. foetus strain used in this work. This work was partially supported by grants from the
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