Acta Tropica 107 (2008) 153–158

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A Mg2+-dependent ecto-phosphatase activity on the external surface ofTrypanosoma rangeli modulated by exogenous inorganic phosphate

Andre L. Fonseca-de-Souza, Claudia Fernanda Dick, Andre Luiz Araujo Dos Santos,Jose Roberto Meyer-Fernandes ∗

Laboratorio de Bioquımica Celular, Instituto de Bioquımica Medica, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro,UFRJ, Cidade Universitaria, Ilha do Fundao, 21941-590 Rio de Janeiro, RJ, Brazil

a r t i c l e i n f o a b s t r a c t

izedenzyp-NP3 mM

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vity. Oed sigable twas n

Article history:Received 27 March 2008Received in revised form 16 May 2008Accepted 22 May 2008Available online 29 May 2008

This work is dedicated to Leopoldo De Meison his 70th birthday.

Keywords:Trypanosoma rangeliEcto-phosphatasePhosphate acquisitionPhosphoserine/threonine phosphatases

In this work, we characterrangeli epimastigotes. Thisp-nitrophenylphosphate (sis was obtained with 0.2substitute Mg2+ as an actdependence on p-NPP conactivity with values of Vma

p-NPP. Mg2+-dependent eusing inhibitors, such as, soMg2+-dependent ecto-phoreversibly in 50% this actiphatase inhibitors, inhibitphosphatase activity waswhile o-phosphotyrosine

exhibit a typical growth curvedependent ecto-phosphatase amaintained at Pi-deprived medimately threefold higher than t

1. Introduction

Trypanosomatids are a group of protozoa that parasitize a largenumber of eukaryotic organisms (D’Alessandro, 1976). In the Try-panosomatidae family, the genus Trypanosoma comprises digeneticflagellates that usually have insects as vectors and infect humanbeings and other animals as hosts (D’Alessandro, 1976; Garcia andAzambuja, 1991). Trypanosoma rangeli is a South American try-panosome able to infect mammals, but apparently unable to elicitpathology in them, although it is detrimental to the insect vec-tor (D’Alessandro, 1976). This parasite co-exists with T. cruzi, theetiologic agent of Chagas’ disease, in the Northern of South Amer-ica, posing some problems for diagnosis (Martınez et al., 1993),

∗ Corresponding author at: Instituto de Bioquımica Medica, Universidade Federaldo Rio de Janeiro, Centro de Ciencias da Saude, bloco H, Cidade Universitaria, Rio deJaneiro, RJ 21941-590, Brazil. Tel.: +55 21 2562 6781; fax: +55 21 2270 8647.

E-mail address: meyer@bioqmed.ufrj.br (J.R. Meyer-Fernandes).

0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2008.05.017

a Mg2+-dependent ecto-phosphatase activity present in live Trypanosomame showed capacity to hydrolyze the artificial substrate for phosphatases,P). At saturating concentration of p-NPP, half-maximal p-NPP hydroly-

Mg2+. Ca2+ had no effect on the basal phosphatase activity, could notand in contrast inhibited the p-NPP hydrolysis stimulated by Mg2+. The

ration showed a normal Michaelis–Menten kinetics for this phosphatase.94 ± 0.36 nmol p-NP × h−1 × 10−7 cells and apparent Km of 1.04 ± 0.16 mMhosphatase activity was stimulated by the alkaline pH range. Experiments

fluoride, sodium orthovanadate and ammonium molybdate, inhibited thetase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibitedkadaic acid and microcystin-LR, specific phosphoserine/threonine phos-nificantly the Mg2+-dependent ecto-phosphatase activity. In addition, thiso recognize as substrates only o-phosphoserine and o-phosphothreonine,ot a good substrate for this phosphatase. Epimastigote forms of T. rangeli

, achieving the stationary phase around fifth or sixth day and the Mg2+-ctivity decreased around 10-fold with the cell growth progression. Cellsium (2 mM Pi) present Mg2+-dependent ecto-phosphatase activity approx-hat maintained at Pi-supplemented medium (50 mM Pi).

© 2008 Elsevier B.V. All rights reserved.

occurring a high immunological cross-reactivity between these twoparasites (Labriola and Cazzulo, 1995).

The life cycle of T. rangeli in the vertebrate host is poorly known.Some evidence suggests that proliferation occurs within monocytes(Osorio et al., 1995). In the invertebrate host, its life cycle is bettercharacterized. After being ingested as trypomastigotes, T. rangelimultiply as epimastigotes in the midgut, invade the hemolymphand hemocytes to continue their growth, and complete their devel-opment in the salivary gland where metacyclogenesis takes place(Hoare, 1972).

The plasma membrane of cells may contain enzymes whoseactive sites face the external medium rather than the cytoplasm.The activities of these enzymes, referred to as ecto-enzymes, canbe measured using intact cells (Gomes et al., 2006; Kiffer-Moreiraet al., 2007; Meyer-Fernandes et al., 1997; Meyer-Fernandes, 2002).Knowledge about interactions between components of the exter-nal surface of the cells and the cellular elements of the host isof obvious importance for the understanding of the complex lifecycle of T. rangeli. Ecto-phosphatases and ecto-kinases have been

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154 A.L. Fonseca-de-Souza et al.

detected in several microorganisms, including protozoa (De Jesuset al., 2002; Dos Passos Lemos et al., 2002; Fernandes et al., 1997;Meyer-Fernandes et al., 1999; Remaley et al., 1984; Sacerdoti-Sierraand Jaffe, 1997), bacteria (Bliska et al., 1991, 1993; Braibant andContent, 2001; Madec et al., 2002) and fungi (Arnold et al., 1986;Bernard et al., 2002; Collopy-Junior et al., 2006; Kiffer-Moreiraet al., 2007; Kneipp et al., 2003, 2004). Several biological rolesfor ecto-phosphatases have been proposed. In fungi these ecto-phosphatases are involved with the infection of the epithelial cells(Collopy-Junior et al., 2006; Kiffer-Moreira et al., 2007; Kneippet al., 2004). In addition, the regulation of the complex inter-actions required for differentiation, proliferation (Bakalara et al.,1995,2000; Meyer-Fernandes et al., 1999) and infection of host cells(Furuya et al., 1998; Martiny et al., 1999; Zhong et al., 1998) ismediated in part by protein phosphorylation/dephosphorylationin higher eukaryotes as well as in trypanosomes.

These enzymes could also provide microbial cells with a sourceof inorganic phosphate (Pi) by hydrolyzing phosphomonoestermetabolites (Bozzo et al., 2004; Braibant and Content, 2001; Kneippet al., 2004; Li et al., 2002) and protect them upon entering themacrophage by suppressing the respiratory burst (Remaley et al.,1984, 1985).

Since ecto-phosphatases may provide cells as a source of Pi, themodulation of expression of these enzymes by exogenous phos-phate content is well known in fungi (Bernard et al., 2002; Braibantand Content, 2001; Kneipp et al., 2004) and plant (Bozzo et al., 2004,2006; Hur et al., 2007; Wang et al., 2008) cells. In this context,the kind of enzyme expressed in medium containing high or lowphosphate content can be the same or different and the differencescorrespond to substrate affinity, hydrolytic ability, inhibitors sensi-tivity, among others (Kneipp et al., 2004). However, the modulationof ecto-phosphatases promoted by Pi content in culture medium inprotozoa has not been investigated.

In this work we have characterized a Mg2+-dependent phos-phatase activity present on the cell surface of T. rangeli, verifyingits substrate specificity, optimum pH and its response to Mg2+, Ca2+

and to inhibitors. In addition, we verified the modulation of thisactivity by exogenous phosphate content. This activity was detectedin intact epimastigote forms of the parasite and will contribute tothe understanding of the physiology and biochemistry of this cell,as well as the event of the interaction parasite–host cell.

2. Materials and methods

2.1. Material

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

2.2. Parasites and growth conditions

Macias strain of T. rangeli (supplied by Dr. Maria AuxiliadoraSousa, from Fiocruz) was maintained at liver infusion tryptose(LIT) medium supplemented with 20% fetal calf serum (Gibco) at28 ± 2 ◦C and subcultivated at 5-day intervals where the parasitesachieve the stationary phase of growth. For the experiments, theparasites were harvested from the culture medium by centrifuga-tion at 1500 × g at 4 ◦C for 10 min and washed twice in a cold buffersolution containing 100 mM sucrose, 20 mM KCl and 50 mM Tris,pH 7.2. Cellular viability was assessed, before and after incubations,by motility and Trypan blue dye exclusion (Leite et al., 2007). ForTrypan staining the cells were incubated in the presence of 0.01%

Tropica 107 (2008) 153–158

Trypan blue for 10 min in the buffer used in each experiment. Theviability was not affected under the conditions employed here.

2.3. Cell proliferation

In the fifth day of culture, parasites were harvested in steriletubes, centrifuged at 1500 × g at 4 ◦C during 10 min and washedtwice with the same cold buffer solution as described before. Thecell density was estimated in a haemocytometric chamber and thegrowth curve was initiated with 1.0 × 106 cells mL−1. The cell pro-liferation was verified every day by the counting of the cell numberin a haemocytometric chamber.

2.4. Ecto-phosphatase activity measurements

Phosphatase activity was determined spectrophotometricallymeasuring the rate of p-nitrophenol (p-NP) production. Intactcells (3 × 107 cells/mL) of T. rangeli were incubated at 25 ◦C for60 min in a reaction mixture (0.5 mL) containing, unless other-wise stated in legends of the figures, 50 mM Tris buffer, pH 7.2,100 mM sucrose, 20 mM KCl and 5 mM p-NPP, as substrate. TheMg2+-dependent phosphatase activity was calculated from thetotal activity, measured in the presence of 5 mM MgCl2 minusthe basal activity measured in the absence of MgCl2. The experi-ments were started by the addition of living cells and terminatedby the addition of 1.0 mL of 1.0N NaOH. After the end of thereaction the same volume of MgCl2 or water were added to thecorrespondent tubes in the absence or in the presence of MgCl2,respectively. The tubes were then centrifuged at 1500 × g for 15 minat 4 ◦C. The phosphatase activity was calculated by subtracting thenonspecific p-NPP hydrolysis measured in the absence of cells.The concentration of p-NP produced in the reaction was mea-sured spectrophotometrically at 405 nm, using a standard curveof p-NP for comparison. In the experiments where other phos-phatase substrates (�-glycerophosphate or phosphoaminoacids)were used, the hydrolytic activities measured under the same con-ditions described above were assayed spectrophotometrically bymeasuring the release of Pi from these substrates, following theFiske and Subbarow (1925) method. The values obtained for p-NPPhydrolysis measured using both methods were exactly the same(Kiffer-Moreira et al., 2007).

2.5. Statistical analysis

All experiments were performed in triplicates, with similarresults obtained from at least three separate cell suspensions.Apparent Km and Vmax values were calculated using a computerizednonlinear regression analysis of the data to the Michaelis–Mentenequation (Saad-Nehme et al., 1997). Data were analyzed statisticallyusing the Student’s t-test. Statistical significance was considered asp < 0.05.

3. Results

3.1. Ecto-phosphatase activity

The measurement of the ecto-phosphatase activity present onthe external surface of T. rangeli cells (Macias strain) was carriedout in living epimastigote forms, maintained at physiological pH(pH 7.2) using p-nitrophenylphosphate (p-NPP) as substrate. Cel-lular viability was assessed before and after incubation by motilityand Trypan blue dye exclusion. The viability was not affected by theconditions employed here (>97%). The time course of p-NPP hydrol-ysis by the phosphatase was linear for at least 60 min (r2 = 0.9939).

A.L. Fonseca-de-Souza et al. / Acta Tropica 107 (2008) 153–158 155

with different cell suspensions.

Fig. 1. Influence of divalent cations on ecto-phosphatase activity of T. rangeli. Theecto-phosphatase activity was measured in a reaction medium containing 50 mMTris buffer, pH 7.2, 20 mM KCl, 100 mM sucrose, 5 mM p-NPP as substrate, in thepresence or in the absence of EDTA and EGTA (1 mM) or divalent cations (5 mMeach). Data are means ± standard errors of three determinations with different cellsuspensions. Asterisk denotes significant differences (p < 0.05) in comparison withcontrol.

Similarly, in assays to determine the influence of cell density, phos-phatase activity measured over 60 min was linear over a nearlyfivefold range of cell density (r2 = 0.9904). The addition of EDTAand EGTA, two divalent metal chelators, did not have effect on thisphosphatase activity (Fig. 1). The addition of 5 mM MgCl2 increasedmore than two times the ecto-phosphatase activity. The addition of5 mM of CaCl2, SrCl2, CdCl2, or NiCl2 to the reaction medium did notincrease the ecto-phosphatase activity (Fig. 1). MgCl2 stimulatedthis phosphatase activity in a dose-dependent manner (Fig. 2). At5 mM p-NPP, half maximum stimulation of p-NPP hydrolysis wasobtained with 0.23 mM MgCl2 and the addition of increasing CaCl2concentrations decreased progressively the Mg2+-dependent phos-phatase activity (Fig. 2).

To exclude the possibility that the observed p-NPP hydrolyzedwas due to secreted soluble enzymes, cells were incubated in theabsence of p-NPP in the absence or in the presence of MgCl2.Subsequently the suspension was centrifuged to remove cells andthe supernatant was assayed for phosphatase activity. This sus-pension failed to show p-NPP hydrolysis (data not shown). Thesedata also rules out the possibility that the phosphatase activ-

Fig. 2. Effects of increasing concentrations of MgCl2 and CaCl2 on the ecto-phosphatase activity of T. rangeli. The ecto-phosphatase activity was measured in areaction medium containing 50 mM Tris buffer, pH 7.2, 20 mM KCl, 100 mM sucrose,5 mM p-NPP as substrate, in the presence of increasing concentrations of MgCl2(closed circles) or 5 mM MgCl2 with increasing concentrations of CaCl2 (open cir-cles). Data are means ± standard errors of three determinations with different cellsuspensions.

Fig. 3. Dependence of p-NPP concentration on Mg2+-dependent ecto-phosphataseactivity of T. rangeli. Ecto-phosphatase activity was measured in a reaction mediumcontaining 50 mM Tris buffer, pH 7.2, 20 mM KCl, 100 mM sucrose, with the additionof increasing concentrations of p-NPP (from 0.1 to 15.0 mM) in the absence or inthe presence of 5 mM MgCl2 and the Mg2+-dependent activity was calculated asdescribed in Section 2. Data are means ± standard errors of three determinations

ity here described could be derived from disrupted T. rangelicells. The dependence on p-NPP concentration presents a nor-mal Michaelis–Menten kinetics for ecto-phosphatase activity of T.rangeli and the kinetic parameters were Vmax of 8.94 ± 0.36 nmolp-NP × h−1 × 10−7 cells and Km of 1.04 ± 0.16 mM (Fig. 3). In the pHrange from 6.8 to 8.4, in which the parasites were viable during allcourse of the reaction, the Mg2+-dependent phosphatase activitywas stimulated in an alkaline range of pH (Fig. 4). Intact epimastig-otes of T. rangeli also recognized phosphorylated aminoacids assubstrates. Interestingly the addition of MgCl2 only stimulated thephosphoserine and phosphothreonine hydrolysis. The phosphoty-rosine hydrolysis was not stimulated by MgCl2 (Fig. 5).

3.2. Effect of phosphatase inhibitors on the phosphatase activity

Different phosphatase inhibitors were tested, and results areshown in Table 1. Ammonium molybdate ((NH4)2MoO4), andsodium orthovanadate (Na3VO4) inhibited the Mg2+-dependent

Fig. 4. Effect of pH on the Mg2+-dependent ecto-phosphatase activity of T. rangeli.Ecto-phosphatase activity was measured in a reaction medium containing 20 mMKCl, 100 mM sucrose, 5 mM p-NPP as substrate and 50 mM Tris buffer, adjusted topH values between 6.8 and 8.4. In this pH range, cells were viable during the courseof reaction. The reactions were performed in the presence and in the absence of5 mM MgCl2 and the Mg2+-dependent activity was calculated as described in Sec-tion 2. Data are means ± standard errors of three determinations with different cellsuspensions.

156 A.L. Fonseca-de-Souza et al. / Acta Tropica 107 (2008) 153–158

Fig. 5. Phosphorylated aminoacids hydrolysis by the ecto-phosphatase activ-ities of T. rangeli. Ecto-phosphatase activities were measured in a reactionmedium containing 50 mM Tris buffer, pH 7.2, 20 mM KCl, 100 mM sucrose and5 mM of each phosphoaminoacids (o-phosphotyrosine, or o-phosphoserine, or o-phosphothreonine), in the presence (open bars) and in the absence of 5 mM MgCl2

(solid bars). Data are means ± standard errors of three determinations with differentcell suspensions. Asterisks denote significant differences (p < 0.05) in comparisonwith the phosphatase activity measured in the absence of MgCl2.

ecto-phosphatase activity in 60% and 77%, respectively andsodium fluoride (NaF) abolished totally the Mg2+-dependent phos-phatase activity. Sodium tartrate, a secreted phosphatase inhibitor(Santos et al., 2002), had no effect on the phosphatase activ-ity. Interestingly levamisole, an alkaline phosphatase inhibitor(Van Belle, 1976), did not inhibit the ecto-phosphatase activity.Okadaic acid and microcystin-LR, specific inhibitors of phosphoser-ine/threonine phosphatases family (Barford et al., 1998; Sommeret al., 2002), inhibited the Mg2+-dependent phosphatase activityin 23% and 83%, respectively. We also tested the Mg2+-dependentecto-phosphatase activity of T. rangeli in the presence of somesubstrates to Mg2+-dependent ecto-nucleotidase activities recentlydescribed in T. rangeli (Fonseca et al., 2006). Adenine nucleotides(ATP, ADP, and 5′AMP) and adenosine were not able to inhibitthe ecto-phosphatase activity (Table 1). These data discard thepossibility that p-NPP hydrolysis could be catalyzed by these

Table 1Effect of different compounds on the Mg2+-dependent ecto-phosphatase activity ofintact cells of T. rangeli

Additions Mg2+-dependent activity (% of control)

Control 100.0 ± 8.0ATP, 10 mM 94.0 ± 4.5ADP, 10 mM 100.0 ± 11.0AMP, 10 mM 96.5 ± 8.0Adenosine, 10 mM 102.0 ± 14.0Levamisole, 1 mM 103.3 ± 9.0Tartrate, 10 mM 100.2 ± 9.0Vanadate, 1 mM 23.0 ± 1.7*NaF, 10 mM 0*Molybdate, 1 mM 41.6 ± 4.1*Pi, 10 mM 53.3 ± 2.0*Okadaic acid, 1 �M 77.3 ± 8.6*Mycrocystin-LR, 1 �М 17.4 ± 1.8*

Ecto-phosphatase activity was measured in a reaction medium containing 50 mMTris buffer, pH 7.2, 20 mM KCl, 100 mM sucrose and 5 mM p-NPP as substrate, in theabsence or in the presence of 5 mM MgCl2 in the presence of various compoundsat the concentrations described in the Table. Control activity (7.57 ± 0.55 nmol p-NP × h−1 × 10−7 cells) was taken as 100% and the standard errors were calculatedfrom the absolute activity values of three experiments with different cell sus-pensions and converted to percentage of the control value. The Mg2+-dependentecto-phosphatase activity was calculated by the difference between the total activ-ity measured in the presence of 5 mM MgCl2 and the basal activity, measured inthe presence of 1 mM EDTA. Asterisks denote significant differences (p < 0.05) aftercomparison with the enzyme activity of control (none addition).

Fig. 6. Effects of different concentrations of inorganic phosphate on the ecto-phosphatase activity of T. rangeli. Cells were incubated for 60 min at 25 ◦C in thesame reaction medium (final volume 0.5 mL) as that described in the legend ofFig. 1, in the absence or in the presence of 5 mM MgCl2, with different concen-trations of inorganic phosphate (Pi) as shown in the abscissa. The Mg2+-dependentphosphatase 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. Dataare means ± standard errors of three determinations with different cell suspensions.Asterisk denotes significant differences (p < 0.05) in comparison with control.

ecto-nucleotidases present on the external surface of T. rangeli.Pi product of reactions catalyzed by phosphatases (De Almeida-Amaral et al., 2006) inhibited reversibly the ecto-phosphataseactivity in a dose-dependent manner (Table 1, Fig. 6).

3.3. Modulation of ecto-phosphatase activity of T. rangeli byexogenous phosphate content

The Mg2+-dependent ecto-phosphatase activity decreased withthe growth phase of T. rangeli. Fig. 7 shows that the Mg2+-dependentecto-phosphatase activity decreases during the time course ofcell growth. The Mg2+-dependent ecto-phosphatase activity is 11-fold higher on the first day than on the fifth day. These datacould be indicating that this enzyme would be important forthe development of T. rangeli. The parasites maintained at Pi-starved culture medium (2 mM Pi) or Pi-supplemented culturemedium (50 mM Pi) were used to determine Mg2+-dependent

Fig. 7. Mg2+-dependent ecto-phosphatase activity (closed circles) during T. rangeliproliferation (open circles). Cells from a culture of T. rangeli at mid-log phase wereharvested, washed twice and seed into fresh medium and grown for the indicatedtimes before being harvested and assayed for ecto-phosphatase activity. At eachtime point, cells were incubated for 60 min at 25 ◦C in the same reaction medium(final volume 0.5 mL) as that described in the legend of Fig. 1, in the absence or in thepresence of 5 mM MgCl2. The Mg2+-dependent phosphatase activity was calculatedfrom the total activity, measured in the presence of 5 mM MgCl2, minus the basalactivity, measured in the absence of MgCl2. Data are means ± standard errors of threedeterminations with different cell suspensions. Cell densities at the time of assayare indicated.

A.L. Fonseca-de-Souza et al. / Acta

Fig. 8. Influence of phosphate content in the culture medium on the Mg2+-dependent ecto-phosphatase activity of T. rangeli. Ecto-phosphatase activity wasmeasured in a reaction medium containing 50 mM Tris buffer, pH 7.2, 20 mM KCl,100 mM sucrose and 5 mM p-NPP as substrate in the absence and in the presenceof 5 mM MgCl2. The Mg2+-dependent phosphatase activity was calculated from thetotal activity, measured in the presence of 5 mM MgCl2, minus the basal activity,measured in the absence of MgCl2. Cells used in this experiment were harvestedfrom the Pi-starved medium (2 mM Pi, black bars) or from the Pi-supplementedmedium (50 mM Pi, white bars) culture media. Data are means ± standard errors ofthree determinations with different cell suspensions.

ecto-phosphatase activity. Ecto-phosphatase activity of epimastig-otes of T. rangeli maintained at Pi-starved culture medium was,approximately, threefold higher than ecto-phosphatase activityfrom epimastigotes grown at Pi-supplemented culture medium(Fig. 8).

4. Discussion

In the present study, the presence of a Mg2+-dependent ecto-phosphatase activity on the external surface of T. rangeli wasdemonstrated. In contrast to other microorganisms (Dutra et al.,2001; Santos et al., 2002; Shnyreva and Egorov, 1990), T. rangelicells do not secrete phosphatases in the extracellular medium. Thisconclusion is based on the fact that the Mg2+-dependent ecto-phosphatase activity was insensitive to sodium tartrate, a secretedphosphatase inhibitor (Santos et al., 2002) (Table 1), and secretedproteins failed to hydrolyze p-NPP. However, our experiments donot rule out the possibility that the phosphatase activity could

be released in vivo once in contact with the insect cells or eventranslocated into these cells, as suggested in the case of Yersiniaphosphatase (Bliska et al., 1993). The most obvious artifact thatcould lead to the hydrolysis of substrates added to intact cells isthe presence of disrupted cells or their disruption in the assayconditions used. The following observations seemingly excludedthis hypothesis: (i) epimastigote forms were viable during the allperiod of incubation, according with the Trypan blue exclusionassays; (ii) the phosphatase activity was linear with time, suggest-ing that eventual cell disruption during the course of incubationwas not adding appreciably to the total activity; (iii) cell disruptionwould also be required for an activity that depended on lysoso-mal enzymes extruded during the assay period which would alsoshow a different kinetics as pointed out in (ii) and also there was nodetectable activity in the supernatants of cells incubated for 60 minin the assay conditions.

The stimulation by Mg2+ of phosphatase activity (Figs. 1 and 2)and its inhibition by millimolar range concentrations of Ca2+

(Fig. 2) could be indicating that in epimastigote form of T. rangelithere is an ecto-phosphatase activity able to hydrolyze phospho-aminoacids under physiological conditions. It has been described

Tropica 107 (2008) 153–158 157

a Mg2+-dependent, Ca2+-inhibitable ecto-phosphatase activity inamastigote form of T. cruzi involved on parasite and host cells inter-action (Meyer-Fernandes et al., 1999).

Reversible phosphorylation of proteins play a major role on theregulation of several cellular processes, including nutrients acqui-sition, cell proliferation, differentiation, migration, and adhesionprocesses (Denu et al., 1996; Hunter, 1995; Tonks and Neel, 1996).The presence of phosphatases regulated by phosphate content inthe culture medium was reported in several prokaryotic organisms(Hullet, 1996; Torriani-Gorini et al., 1994), and in fungi (Bernardet al., 2002; Kneipp et al., 2004; Ogawa et al., 2000), but not inprotozoa parasites. In S. cerevisiae, transcription of genes encodingacid and alkaline phosphatases and Pi transporter are coordinatelyrepressed and derepressed depending on the Pi concentration inthe culture medium (Oshima et al., 1996). The regulation of thisadaptative response is very complex, involving several genes thatsignal Pi starvation (Ogawa et al., 2000). Most of the phosphatasessynthesized under Pi-limiting conditions are located extracellularlyor are associated with the plasma membrane (Metzenberg, 1979).

The Mg2+-dependent ecto-phosphatase activity described heredecreased concomitantly with progression of cell growth of T.rangeli (Fig. 7). This phosphatase was also strongly and reversiblyinhibited by Pi and cultivation of T. rangeli at low Pi concentra-tions resulted in the generation of protozoan cells expressing anecto-phosphatase, approximately, threefold higher than expressedby T. rangeli cultivated in the presence of high Pi concentrations.It remains to be elucidated if the depletion of phosphate fromthe culture medium induced the expression of a different ecto-phosphatase activity as described in the fungi Fonsecaea pedrosoi(Kneipp et al., 2004).

Acknowledgements

We would like to thank Dr. Maria Auxiliadora, from the Try-panosomatid collection Fiocruz, by supplement of the T. rangeli.We would like also to thank Mr. Fabiano Ferreira Esteves and Ms.Rosangela Rosa de Araujo for the excellent technical assistance.This work was supported by grants from the Brazilian AgenciesConselho Nacional de Desenvolvimento Cientıfico e Tecnologico(CNPq), Coordenacao de Aperfeicoamento de Pessoal de Nıvel Supe-rior (CAPES) and Fundacao de Amparo a Pesquisa do Estado do Riode Janeiro (FAPERJ).

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