A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms

Veterinary Parasitology 118 (2003) 19–28

A metallo phosphatase activity present on the surface of Trypanosoma brucei procyclic forms Eloise Cedro Fernandes a , José Mauro Granjeiro b , Hiroshi Aoyama b , Fábio Vasconcelos Fonseca c , José Roberto Meyer-Fernandes c,∗ , An´ıbal Eugˆenio Vercesi d a

c

Centro de Genética, Biologia Molecular e Fitoqu´ımica-Instituto, Agronomico de Campinas, 13075-630 Campinas, SP, Brazil b Departamento de Bioqu´ımica, Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil Departamento de Bioqu´ımica Médica, ICB, Universidade Federal do Rio de Janeiro (UFRJ), CCS, Bloco H, Cidade Universitária, Ilha do Fundão, 21541-590 Rio de Janeiro, RJ, Brazil d Departamento de Patologia Cl´ınica (NMCE), Universidade Estadual de Campinas, 13083-970 Campinas, SP, Brazil Received 30 October 2002; received in revised form 26 September 2003; accepted 28 September 2003

Abstract In this work, we describe how living cells of Trypanosoma brucei procyclic forms were able to hydrolyze extracellular p-nitrophenylphosphate (pNPP). These intact parasites, which had their viability determined by motility and the Trypan blue method, presented a low level of pNPP hydrolysis in the absence of any divalent metal (0.72 ± 0.07 nmol pNP/mg min). Interestingly, in the presence of 5 mM MgCl2 , ectophosphatase activity of 1.91 ± 0.21 nmol pNP/mg min was observed. The ectophosphatase activity was also stimulated by MnCl2 , CoCl2 and CuCl2 but not by CaCl2 and CdCl2 and was inhibited by ZnCl2 . The addition of Mg2+ , Mn2+ , Co2+ and Cu2+ to extracellular medium increased the ectophosphatase activity in a dose-dependent manner. At 5 mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39 ± 0.05 mM MgCl2 , 0.33 ± 0.03 mM MnCl2 , 1.63 ± 0.12 mM CoCl2 and 2.04 ± 0.33 mM CuCl2 . In the absence of any divalent metal (basal activity) the apparent Km for pNPP was 0.66 ± 0.09 mM, while at saturating MgCl2 concentrations the corresponding apparent Km for pNPP for Mg2+ -stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.27 ± 0.03 mM. The Mg2+ -stimulated pNPP hydrolysis was strongly inhibited by ZnCl2 and vanadate, while the

Abbreviations: pNPP, p-nitrophenylphosphate; pNP, p-nitrophenol; Tris, (tris[hydroxymethyl]aminomethane) Corresponding author. Tel.: +55-21-590-4545; fax: +55-21-2270-8647l. E-mail address: [email protected] (J.R. Meyer-Fernandes). ∗

0304-4017/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2003.09.012

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metal-independent basal phosphatase activity was less inhibited by these phosphotyrosyl phosphatase inhibitors. © 2003 Elsevier B.V. All rights reserved. Keywords: Trypanosoma brucei; Ectophosphatase; Phosphotyrosine

1. Introduction Trypanosoma brucei, the parasitic protozoan agent of Nagana in the African cattle, is transmitted between mammals by the tsetse fly. Its life cycle includes several distinct nonreplicative and proliferative stages in both the mammalian host and the insect vector. In addition to biochemical and morphological modifications, the differentiation from the mammalian bloodstream form to the insect procyclic form is accompanied by important changes in protein composition of the cell surface (Rolin et al., 1996). The plasma membrane of cells may contain enzymes whose active sites face the external medium rather than the cytoplasm. The activities of these enzymes, referred to as ectoenzymes, can be measured using intact cells (Meyer-Fernandes et al., 1997; Peres-Sampaio et al., 2001; Jesus et al., 2002; Lemos et al., 2002; Meyer-Fernandes, 2002). To establish that an enzyme is indeed an ectoenzyme, some criteria may be used (Plesner, 1995): (1) the enzyme acts on extracellular substrate; (2) cellular integrity is maintained; (3) the products are released extracellularly; (4) the enzyme activity is not released to the extracellular; and (5) the enzyme activity can be modified by nonpenetrating reagents. Knowledge about interactions between components of the external surface of the T. brucei brucei and the cellular elements of the host is of obvious importance for the understanding of the complex pathology of Nagana disease. The presence of membrane-bound acid phosphatases has been reported in Trypanosoma rhodesiense (McLaughlin, 1986), Trypanosoma congolense (Tosomba et al., 1996), T. brucei (Fernandes et al., 1997; Bakalara et al., 2000), Trypanosoma cruzi (Meyer-Fernandes et al., 1999), and some Leishmania species (Lovelace and Gottlieb, 1986; Vannier-Santos et al., 1995; Martiny et al., 1996). In Leishmania donovani acid phosphatase activity was suggested as a marker of virulence (Katakura and Kobayashi, 1988; Singla et al., 1992). Reversible phosphorylation of proteins is recognized as a major mechanism for the control of intracellular events in eukaryotic cells. Phosphorylation–dephosphorylation of serine, threonine, and tyrosine residues triggers conformational changes in proteins that alter their biological properties (Cohen, 1989; Hunter, 1995). The regulation of the complex interactions required for differentiation and proliferation is mediated in part by protein phosphorylation in higher eukaryotes (Hunter, 1995), as well as in Trypanosomes (Parsons et al., 1993). Such phosphorylation is reversible, and several phosphatases active towards phosphotyrosyl [Tyr(P)]-proteins (Swarup et al., 1981) have been described as acid (Lau et al., 1989) and alkaline phosphatases (Swarup et al., 1981; Lau et al., 1989). In various tissues and cells the presence of phosphotyrosyl protein phosphatases, which are also active toward low molecular weight, nonprotein phosphoesters such as alkyl and aryl phosphates, including p-nitrophenylphosphate, has been described (Lau et al., 1989; Zhang, 1995; Montserat et al., 1996). The presence of protein tyrosine phosphatase activities in L.

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donovani (Cool and Blum, 1993), T. brucei (Bakalara et al., 1995) and T. cruzi (Bakalara et al., 1995) has been demonstrated. More recently the presence of ecto-protein phosphatase activities in T. cruzi (Furuya et al., 1998) and T. brucei (Bakalara et al., 2000) has also been demonstrated; however, the modulation promoted by divalent cations has not been investigated. In this work, we show the presence of a metallo phosphatase activity and a phosphatase activity independent of divalent cations on the cell surface of T. brucei brucei procyclic forms that can be distinguished by their substrate affinity as well as response to divalent cations and to inhibitors.

2. Materials and methods 2.1. Parasite culture T. brucei brucei procyclic forms (ILTar 1 procyclics) were grown at 28 ◦ C in SDM-79 medium (Cunningham, 1977) supplemented with hemin (7.5 mg/ml) and 10% fetal bovine serum. Two to 3 days after inoculation, cells were collected by centrifugation, washed twice and kept in 50 mM Tris–HCl, pH 7.2, 20 mM KCl and 100 mM sucrose. Cellular viability was assessed before and after incubations by mobility and Trypan blue methods (Dutra et al., 1998). The viability was not affected under the conditions employed here. The protein concentration was determined by biuret assay (Gornall et al., 1949). 2.2. Phosphatase measurements Unless otherwise specified, pNPP hydrolysis was measured as follows: the reaction mixtures (0.5 ml) contained 10 mM pNPP, 50 mM Tris–HCl, pH 7.2, 100 mM sucrose, 20 mM KCl, and 1.0 mg of cell protein. Reactions were initiated by the addition of intact cells (1 mg of protein/ml which corresponds to 1.2 × 108 cells/ml) to the reaction mixtures, incubated at 28 ◦ C with gentle shaking (40 oscillations/min) and terminated after 60 min by the addition of 1 ml of 1 N NaOH. The tubes were then centrifuged at 1000 × g for 20 min at 4 ◦ C. The phosphatase activity was calculated by subtracting the nonspecific pNPP hydrolysis measured in the absence of parasites. The reaction was spectrophotometrically determined at 425 nm using an extinction coefficient of 14.3 × 103 M−1 cm−1 (Rodrigues et al., 1999). Specific activity is expressed as nmol of p-nitrophenol (pNP) released/min/mg of protein. 2.3. Statistical analysis All experiments were performed in triplicate, with similar results obtained in at least three separate cell suspensions. Apparent Km for pNPP and Vmax values were calculated using an iterative nonlinear regression analysis of the data to the Michaelis–Menten equation (Saad-Nehme et al., 1997). Statistical significance was determined by Student’s t test. Significance was considered as P < 0.05.

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2.4. Chemicals All reagents were purchased from E. Merck (D-6100 Darmstadt, Germany) or Sigma Chemical Co. (St. Louis, MO). Distilled water was deionized using a MilliQ system of resins (Millipore Corp., Bedford, MA) and was used in the preparation of all solutions.

3. Results In this work, we show the presence of a metal-stimulated ectophosphatase activity present on the external surface of the procyclic form. Cellular integrity and viability were assessed before and after the reactions, by motility and cell dye exclusion (Dutra et al., 1998). The integrity of the cells was not affected by any conditions used in the assays. The time course of ectophosphatase activity was linear for at least 60 min (r 2 = 0.9993). Similarly, in assays to determine the influence of cell density, the phosphatase activity measured over 60 min was linear over a nearly 6-fold range of cell density (r 2 = 0.9989). The ectophosphatase activity was stimulated by MgCl2 , MnCl2 , CoCl2 and CuCl2 but not by CaCl2 and CdCl2 . Under similar conditions, ZnCl2 was an inhibitor (Fig. 1). The addition of Mg2+ , Mn2+ , Co2+ and Cu2+ to extracellular medium increased the ectophosphatase activity in a dose-dependent manner (Fig. 2). At 5 mM pNPP, half-maximal stimulation of pNPP hydrolysis was obtained with 0.39 ± 0.05 mM MgCl2 (Fig. 2A), 0.33 ± 0.03 mM MnCl2 (Fig. 2B), 1.63 ± 0.12 mM CoCl2 (Fig. 2C) and 2.04 ± 0.33 mM CuCl2 (Fig. 2D).

Phosphatase Activity nmol p-NP . mg-1 . min-1

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2.0

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Fig. 1. Influence of different divalent cations on the ectophosphatase activity of intact cells of T. brucei. Cells were incubated for 1 h at 28 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in the Section 2, with the addition of 3 mM of each divalent cation. Data are means ± S.E. of three determinations with different cell suspensions.

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(A)

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Phosphatase Activity nmol p-NP . mg-1 . min-1

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Divalent Cation (mM) Fig. 2. Influence of different cation concentrations on the ectophosphatase activity of intact cells of T. brucei. Cells were incubated for 1 h at 28 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in the Section 2, with the addition of increasing concentrations of MgCl2 (A), MnCl2 (B), CoCl2 (C) or CuCl2 (D). Data are means ± S.E. of three determinations with different cell suspensions.

To check whether the observed metal-stimulated phosphatase activity could be the result of secreted soluble enzymes, we incubated the cells in a reaction mixture containing 5 mM MgCl2 without pNPP. Subsequently, the cells were removed by centrifugation and the supernatant was assayed for phosphatase activity. This supernatant failed to show pNPP hydrolysis (data not shown). These data also rule out the possibility that the metal-stimulated phosphatase activity here described could be from lysed T. brucei. In the pH range from 6.5 to 8.5, in which the T. brucei was viable, the basal phosphatase activity (measured in the absence of any divalent metal), was inactivated by the increase of pH (Fig. 3, open circle), while the Mg-stimulated phosphatase activity (difference between total (measured in the presence of 5 mM MgCl2 ) minus the basal activity) was weakly inactivated (Fig. 3, closed circle). The dependence on pNPP concentration shows a normal Michaelis–Menten kinetics for both phosphatase activities, and the values of Vmax and apparent Km for pNPP were 0.72 ± 0.07 nmol pNP and 0.66 ± 0.09 mM, respectively, for basal phosphatase activity (Fig. 4A) and 1.26 ± 0.16 nmol pNP and 0.27 ± 0.03 mM, respectively, for the Mg-stimulated phosphatase activity (Fig. 4B). As shown in Fig. 5 the Mg2+ -dependent phosphatase activity was strongly inhibited by micromolar concentrations of ZnCl2 (Ki = 203.8±23.3 ␮M; Fig. 5A, closed circle) and vanadate (Ki = 12.6±1.4 ␮M; Fig. 5B, closed circle), while the Mg2+ -insensitive phosphatase activity was less inhibited by ZnCl2 (Ki = 504.2±61.7 ␮M; Fig. 5A, open circle) and vanadate (Ki = 26.8±3.2 ␮M; Fig. 5B, open circle).

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Phosphatase Activity nmol p-NP . mg-1 . min-1

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Phosphatase Activity nmol p-NP . mg-1 . min-1

Fig. 3. Effect of pH on the ectophosphatase activities of intact cells of T. brucei. Cells were incubated for 1 h at 28 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in the Section 2, adjusted to pH values between 6.5 and 8.5 with HCl and Tris, in the absence (䊊) or in the presence of MgCl2 (䊉).The metal-stimulated ectophosphatase 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 . In this pH range the cells were viable throughout the course of the experiments. Data are means ± S.E. of three determinations with different cell suspensions.

1.50

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p-NPP (mM) Fig. 4. Dependence of pNPP concentrations of intact cells of T. brucei ectophosphatase activities. Cells were incubated for 1 h at 28 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in the Section 2, in the absence (A) or in the presence of MgCl2 (B). The metal-stimulated ectophosphatase 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.

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Phosphatase Activity (%)

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Fig. 5. Inhibition of T. brucei ectophosphatases activities by phosphotyrosine phosphatase inhibitors. Cells were incubated for 1 h at 28 ◦ C in the same reaction medium (final volume: 0.5 ml) as that described in the Section 2, with increasing concentration of ZnCl2 (A) and vanadate (B) in the absence (䊊) or in the presence of MgCl2 (䊉).The metal-stimulated ectophosphatase 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.

4. Discussion Little is known about the functionality of membrane-bound enzymes in living cells and their possible role in the process of host–parasite interactions. The detection of cell surface located phosphatase activity is particularly interesting due to its possible role in cell–cell interaction or reception and transduction of external stimuli (Vannier-Santos et al., 1995; Martiny et al., 1996, 1999). Cellular responses to extracellular stimuli (e.g., parasite adhesion) can evoke signaling pathways including protein phosphorylation–dephosphorylation. It was shown that Leishmania acid phosphatases can regulate parasite binding to macrophages (Vannier-Santos et al., 1995; Martiny et al., 1996). Ectophosphatase activities not stimulated by divalent cation have also been detected in some parasitic protozoa Herptomonas muscarum muscarum (Dutra et al., 1998, 2001), Phytomonas françai (Dutra et al., 2000) and T. brucei (Fernandes et al., 1997; Bakalara et al., 2000). Previously we described an ectophosphatase activity independent of divalent cations present on the external surface of intact T. brucei procyclic forms, able to hydrolyze 0.72 nmol pNPP/mg min) (Fernandes et al., 1997). In this work, we demonstrated that in external cell surface of T. brucei procyclic forms there are two phosphatase activities, distinguished by their substrate affinity (Fig. 4) and their responses to inhibitor and activator cations (Figs. 1–3 and 5). The metal-stimulated phosphatase activity present on the external surface of T. brucei is present in the procyclic form and absent in the bloodstream form of T. brucei (Bakalara et al., 2000; Fernandes et al., 2003). The high sensitivity to ZnCl2 (Fig. 5, panel A) and vanadate (Fig. 5, panel B), two known potent and specific phosphotyrosyl protein phosphatase inhibitors (Swarup et al., 1981; Lau et al., 1989), suggest that this Mg2+ -dependent phosphatase has similarities with the Mg-stimulated ecto-protein phosphatase present in other parasitic protozoa like T. cruzi

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(Meyer-Fernandes et al., 1999) and might dephosphorylate phosphoproteins phosphorylated in tyrosine and serine residues on host cells. The reason for the incomplete inhibition of the Mg2+ -dependent phosphatase activity by ZnCl2 (Fig. 5, panel A) and vanadate (Fig. 5, panel B) remains unclear. It is possible that the supposed selective action of these inhibitors depends on the catalytic mechanism of the enzymes, substrate specificity and association with possible specific regulatory subunits. Other protein phosphatases, such as the receptor protein tyrosine phosphatase (RPTP), were shown to have an important role in the process of cell–cell adhesion (Fischer et al., 1991; Gebbink et al., 1993). The biological role of the ectophosphatases present in protozoa parasites is still unknown. However, it has been suggested that ectophosphorylation regulation in ectodomains of functionally important surface proteins and/or soluble external substrates are involved in processes such as inhibition of cell growth (Friedberg et al., 1995; Bakalara et al., 2000) and parasite–host interactions (Bakalara et al., 2000). The biological role of the metal-stimulated ectophosphatase activity present on the external surface of the T. brucei procyclic form remains to be elucidated.

Acknowledgements We thank Dr. Alicia J. Kowaltowski for critical reading of the manuscript. This work was partially supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de N´ıvel Superior (CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPERJ), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPESP) and Programa de Núcleos de Excelˆencia (PRONEX). References Bakalara, N., Seyfang, A., Davis, C., Baltz, T., 1995. Characterization of a life-cycle-stage-regulated protein tyrosine phosphatase in Trypanosoma brucei. Eur. J. Biochem. 234, 871–877. Bakalara, N., Santarelli, X., Davis, C., Baltz, T., 2000. Purification, cloning, and characterization of an acidic ectoprotein phosphatase differentially expressed in the infectious bloodstream form of Trypanosoma brucei. J. Biol. Chem. 275, 8863–8871. Cohen, P., 1989. The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58, 453–508. Cool, D.E., Blum, J.J., 1993. Protein tyrosine phosphatase activity in Leishmania donovani. Mol. Cell. Biochem. 127, 143–149. Cunningham, I., 1977. New culture medium for maintenance of tsetse tissues and growth of trypanosomatids. J. Protozool. 24, 325–329. 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 ectophosphatase activity of Herpetomonas muscarum muscarum inhibited by platelet-activating factor. Biochem. Biophys. Res. Commun. 253, 164–169. Dutra, P.M.L., Rodrigues, C.O., Romeiro, A., Grillo, L.A.M., Dias, F.A., Attias, M., De Souza, W., Lopes, A.H.C.S., Meyer-Fernandes, J.R., 2000. Characterization of ectophosphatase activities in trypanosomatid parasites of plants. Phytopathology 90, 1032–1038. Dutra, P.M.L., Dias, F.A., Rodrigues, C.O., Romeiro, A., Attias, M., De Souza, W., Lopes, A.H.C.S., MeyerFernandes, J.R., 2001. Platelet-activating factor modulates a secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. Curr. Microbiol. 43, 288–292. Fernandes, E.C., Meyer-Fernandes, J.R., Silva-Neto, M.A.C., Vercesi, A.E., 1997. Trypanosoma brucei: ectophosphatase present on the surface of intact procyclic forms. Z. Naturforsch. 52c, 351–358.

E.C. Fernandes et al. / Veterinary Parasitology 118 (2003) 19–28

27

Fernandes, E.C., Granjeiro, J.M., Taga, E.M., Meyer-Fernandes, J.R., Aoyama, H., 2003. Phosphatase activity characterization on the surface of intact bloodstream forms of Trypanosoma brucei. FEMS Microbiol. Lett. 220, 197–206. Fischer, E.H., Charbonneau, H., Tonks, N.K., 1991. Protein tyrosine phosphatase: a diverse family of intracellular and transmembrane enzymes. Science 253, 401–406. Friedberg, I., Belzer, I., Ogel-Plez, O., Kuebler, D., 1995. Activation of cell growth inhibitor by ectoprotein kinase-mediated phosphorylation transformed mouse fibroblasts. J. Biol. Chem. 270, 20560–20567. 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 stages. Mol. Biochem. Parasitol. 92, 339–348. Gebbink, M.F.B.G., Zondag, G.C.M., Wubbolts, R.W., Beijersberge, R.L., Van Etten, I., Moolenaar, W.H., 1993. Cell–cell adhesion mediated by a receptor-like protein tyrosine phosphatase. J. Biol. Chem. 268, 16101–16104. Gornall, A.G., Bardawill, C.J., David, M.M., 1949. Determination of serum proteins by means of the biuret reactions. J. Biol. Chem. 177, 751–766. Hunter, T., 1995. Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225–236. Jesus, J.B., Lopes, A.H.C.S., Meyer-Fernandes, J.R., 2002. Characterization of an ecto-ATPase of Tritrichomonas foetus. Vet. Parasitol. 103, 29–42. Katakura, K., Kobayashi, A., 1988. Acid phosphatase activity of virulent and avirulent clones of Leishmania donovani promastigotes. Infect. Immun. 56, 2856–2860. Lau, K.H., Farley, J.R., Baylink, D.J., 1989. Phosphotyrosyl protein phosphatases. Biochem. J. 257, 23–36. Lemos, A.P., Souza, A.L.F., Pinheiro, A.A.S., Berrˆedo-Pinho, M., Meyer-Fernandes, J.R., 2002. Ecto-phosphatase activity on the cell surface of Crithidia deanei. Z. Naturforsch. 57, 500–505. Lovelace, J.K., Gottlieb, M., 1986. Comparison of extracellular acid phosphatases from various isolates of Leishmania. Am. J. Trop. Med. Hyg. 35, 1121–1128. Martiny, A., Vannier-Santos, M.A., Borges, V.M., Meyer-Fernandes, J.R., Asseruy, 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. McLaughlin, J., 1986. The association of distinct acid phosphatases with the flagella pocket and surface membrane fractions obtained from bloodstream forms of T. rhodesiense. Mol. Cell Biochem. 70, 177–184. Meyer-Fernandes, J.R., 2002. Ecto-ATPases in protozoa parasites: looking for a function. Parasitol. Int. 51, 299– 303. 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. Ectophosphatase activities on the cell surface of the amastigotes forms of Trypanosoma cruzi. Z. Naturforsch. 54c, 977–984. Montserat, J., Chen, L., Lawrence, D.S., Zhang, Z.-Y., 1996. Potent low molecular weight substrates for protein-tyrosine phosphatase. J. Biol. Chem. 271, 7868–7872. Parsons, M., Valentine, M., Carter, V., 1993. Protein kinases in divergent eukaryotes: identification of protein kinase activities regulated during trypanosome development. Proc. Natl. Acad. Sci. USA 90, 2656–2660. Peres-Sampaio, C.E., Thorp Palumbo, S., Meyer-Fernandes, J.R., 2001. An ecto-ATPase activity present in Leishmania tropica stimulated by dextran sulfate. Z. Naturforsch. 56, 820–825. Plesner, L., 1995. Ecto-ATPases: identities and functions. Int. Rev. Cytol. 158, 141–214. Rodrigues, C.O., Dutra, P.M.L., Barros, F.S., Souto-Padrón, T., Meyer-Fernandes, J.R., Lopes, A.H.C.S., 1999. Platelet-activating factor induction of secreted phosphatase activity in Trypanosma cruzi. Biochem. Biophys. Res. Commun. 266, 36–42. Rolin, S., Hanocq-Quertier, J., Paturiaux-Hanocq, F., Nolan, D., Salmon, D., Webb, H., Carrington, M., Voorheis, P., Pays, E., 1996. Simultaneous but independent activation of adenylate cyclase and glycosylphosphadylinositol-phospholipase C under stress conditions in Trypanosoma brucei. J. Biol. Chem. 271, 10852–10884.

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Saad-Nehme, J., Bezerra, A.L., Fornells, L.A.M., Silva, J.L., Meyer-Fernandes, J.R., 1997. A contribution of the mitochondrial adenosinetriphosphatase inhibitor protein to the thermal stability of the F0 F1 ATPase complex. Z. Naturforsch. 52c, 459–465. Singla, N., Khuller, G.K., Vinayak, V.K., 1992. Acid phosphatase activity of promastigotes of L. donovani: a marker of virulence. FEMS Microbiol. Lett. 94, 221–226. Swarup, G., Cohen, S., Garbers, D.L., 1981. Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J. Biol. Chem. 256, 8197–8201. Tosomba, O.M., Coetzer, T.H.T., Lonsdale-Eccles, J.D., 1996. Localization of acid phosphatase activity on the surface of bloodstream forms of Trypanosoma congolense. Exp. Parasitol. 84, 429–438. Vannier-Santos, M.A., Martiny, A., Meyer-Fernandes, J.R., De Souza, W., 1995. Leishmanial protein kinase C modulates host cell infection via secreted acid phosphatase. Eur. J. Cell. Biol. 67, 112–119. Zhang, Z.-Y., 1995. Are protein-tyrosine phosphatases specific for phosphotyrosine. J. Biol. Chem. 270, 16052– 16055.