Experimental Parasitology 137 (2014) 8–13

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Experimental Parasitology

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Leishmania amazonensis: Characterization of an ecto-pyrophosphataseactivity

0014-4894/$ - see front matter � 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.exppara.2013.11.008

Abbreviations: Ecto-PPase, ecto-pyrophosphatase; Pi, inorganic phosphate; PPi,pyrophosphate; PPase, pyrophosphatase; sPPase, soluble pyrophosphatase.⇑ Corresponding author at: Instituto de Bioquímica Médica, Universidade Federal

do Rio de Janeiro, CCS, Bloco H, Cidade Universitária, 21941-590 Rio de Janeiro, RJ,Brazil. Fax: +55 21 2270 8647.

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

Anita Leocadio Freitas-Mesquita a,b, André Luiz Fonseca-de-Souza c, José Roberto Meyer-Fernandes a,b,⇑a Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, RJ, Brazilb Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagem, 21941-590 Rio de Janeiro, RJ, Brazilc Laboratório de Terapia e Fisiologia Celular e Molecular, Centro Universitário Estadual da Zona Oeste, 23070-200 Rio de Janeiro, RJ, Brazil

h i g h l i g h t s

� Leishmania amazonensis possesses anecto-pyrophosphatase activity.� The ecto-pyrophosphatase activity is

stimulated by MgCl2 but not byMnCl2.� The ecto-pyrophosphatase activity is

increased at alkaline pHs.� The ecto-pyrophosphatase is not

inhibited by classical phosphataseinhibitors.� The ecto-pyrophosphatase activity is

likely involved in cell growth.

g r a p h i c a l a b s t r a c t

a r t i c l e i n f o

Article history:Received 13 September 2013Received in revised form 22 November 2013Accepted 27 November 2013Available online 6 December 2013

Keywords:Leishmania amazonensisEcto-pyrophosphataseInorganic pyrophosphate

a b s t r a c t

Several ecto-enzymatic activities have been described in the plasma membrane of the protozoan Leish-mania amazonensis, which is the major etiological agent of diffuse cutaneous leishmaniasis in SouthAmerica. These enzymes, including ecto-phosphatases, contribute to the survival of the parasite by par-ticipating in phosphate metabolism. This work identifies and characterizes the extracellular hydrolysis ofinorganic pyrophosphate related to an ecto-pyrophosphatase activity of the promastigote form of L.amazonensis. This ecto-pyrophosphatase activity is insensitive to MnCl2 but is strongly stimulated byMgCl2. This stimulation was not observed during the hydrolysis of p-nitrophenyl phosphate (p-NPP) orb-glycerophosphate, two substrates for different ecto-phosphatases present in the L. amazonensis plasmamembrane. Furthermore, extracellular PPi hydrolysis is more efficient at alkaline pHs, while p-NPPhydrolysis occurs mainly at acidic pHs. These results led us to conclude that extracellular PPi ishydrolyzed not by non-specific ecto-phosphatases but rather by a genuine ecto-pyrophosphatase. Inthe presence of 5 mM MgCl2, the ecto-pyrophosphatase activity from L. amazonensis is sensitive to micro-molar concentrations of NaF and millimolar concentrations of CaCl2. Moreover, this activity is signifi-cantly higher during the first days of L. amazonensis culture, which suggests a possible role for thisenzyme in parasite growth.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Leishmaniasis is a major insect-borne disease in developingcountries, and 350 million people in 88 countries worldwide liveat risk of developing one of the many forms of the disease(Vannier-Santos et al., 2002). Depending on the species ofLeishmania, the disease can manifest in a cutaneous or visceral

A.L. Freitas-Mesquita et al. / Experimental Parasitology 137 (2014) 8–13 9

form (Kaye and Scott, 2011; Lopes et al., 2010). Leishmania ama-zonensis is the major etiological agent of diffuse cutaneous leish-maniasis in South America (Reithinger et al., 2007; Silveira et al.,2009).

In the Leishmania life cycle, the motile promastigote form istransmitted from the sandfly vector to a mammalian host duringa blood meal. Inside vertebrate host macrophages, the parasitesdifferentiate into a nonmotile amastigote form (Vannier-Santoset al., 2002; Burchmore et al., 2003). Inside both insect and mam-malian hosts, the parasites must survive in several microenviron-ments with differing pH (Sen et al., 2009) and nutrientavailability (Vannier-Santos et al., 2002; Gomes et al., 2011). Theecto-enzymes present in the plasma membrane of the parasitesmay play a role in survival by providing essential nutrients frommolecules in the external medium (De Almeida-Amaral et al.,2006; Vieira et al., 2011; Paletta-Silva et al., 2011; Giarola et al.,2013).

Inorganic pyrophosphate (PPi) is a product of cellular metabo-lism that can be hydrolyzed by cytosolic or membrane-boundpyrophosphatases (PPases) to generate two molecules of inorganicphosphate (Pi) (López-Marqués et al., 2004). Soluble pyrophospha-tases (sPPases) are essential and ubiquitous metal-dependent en-zymes capable of providing a thermodynamic pull for manyimportant reactions, such as the biosynthesis of DNA and RNA. sPP-ases comprise two non-homologous families; family I sPPases arefound in all type of organisms, and family II sPPases are found onlyin bacteria and archaea (Hoelzle et al., 2010). Unlike the well-char-acterized family I sPPases, family II sPPases prefer Mn2+ to Mg2+ asa cofactor and are not inhibited by Ca2+ (Zyryanov et al., 2004).

Membrane-bound PPases have no sequence similarity to sPPas-es (Baltscheffsky et al., 1999); however, these PPases are capable ofutilizing the energy released from the cleavage of a phosphoanhy-dride bond to establish a transmembrane ionic gradient. H+PPasesare usually classified according to their sensitivity to K+ and havebeen described in the vacuoles of archaea, bacteria, protists andhigher plants (Pérez-Castiñeira et al., 2001). H+PPases have neverbeen identified in mammals, so they are considered potential ther-apeutic targets (Sen et al., 2009). Several membrane-associatedPPases are also found in animal and yeast mitochondria, but theircapacity for ion transport has not been described (Baykov et al.,1993).

In protozoan parasites, sPPases have been described in Trypan-osoma brucei (Lemercier et al., 2004) and L. amazonensis (Espiauet al., 2006) as key enzymes in the regulation of polyphosphatemetabolism in the acidocalcisome. Other vacuolar H+PPases havebeen reported in Trypanosoma cruzi (Scott et al., 1998), T. brucei(Rodrigues et al., 1999a), Leishmania donovani (Rodrigues et al.,1999b), Plasmodium falciparum (Luo et al., 1999) and Toxoplasmagondii (Rodrigues et al., 2000).

Although most of the PPases characterized in protozoa arefound in the acidocalcisome, H+PPase activity in the plasmamembrane of L. donovani promastigote and amastigote formswas recently identified (Sen et al., 2009). In this study, we iden-tified a pyrophosphatase activity found at the external surface ofthe plasma membrane of L. amazonensis promastigotes. Thisecto-pyrophosphatase (ecto-PPase) activity was characterizedwith respect to several biochemical parameters, such as metalrequirements, response to pH variation and sensitivity to differ-ent inhibitors.

2. Materials and methods

2.1. Materials

All reagents were purchased from E. Merck (Darmstadt,Germany) or Sigma Chemical Co. (St. Louis, MO, USA). The water

used in the preparation of all solutions was filtered through afour-stage Milli-Q filter (Millipore Corp., Bedford, MA, USA).

2.2. Parasites and growth conditions

The MHOM/BR/75/Josefa strain of L. amazonensis was usedthroughout this study. This strain was isolated from a human caseof diffuse cutaneous leishmaniasis in Brazil by Dr. Cuba-Cuba (Uni-versidade de Brasília, Brazil) and has been maintained within ourlaboratory in axenic culture as well as through hamster footpadinoculation. Promastigotes, cultured for long periods in axenicmedium, were cultured in Warren’s medium supplemented with10% (v/v) heat-inactivated fetal bovine serum at 22 �C. For theexperiments in this study, the parasites were harvested from theculture medium (seven days after inoculation) by centrifugationat 1500�g at 4 �C for 10 min and washed twice in a cold buffersolution containing 116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucoseand 50.0 mM HEPES buffer (pH 7.4). Cellular viability was assessedbefore and after incubations by motility and Trypan blue dyeexclusion (de Souza Leite et al., 2007). For Trypan staining, the cellswere incubated in the presence of 0.01% Trypan blue for 10 min inthe buffer used for each experiment. Viability was not affected un-der the conditions employed here.

2.3. Ecto-pyrophosphatase activity measurements

Pyrophosphatase activity was determined by using PPi as thesubstrate and measuring the rate of Pi production. Living prom-astigotes of L. amazonensis (1.0 � 107 cells/ml) were incubated at25 �C for 60 min in 0.5 ml of a reaction mixture containing116.0 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 5.0 mM MgCl2,50.0 mM HEPES buffer (pH 7.4) and 1 mM PPi as the substrate.The pyrophosphatase activity was calculated by subtracting thenonspecific PPi hydrolysis measured in the absence of parasitecells. To determine the concentration of released Pi, the productof PPi hydrolysis, 0.5 ml aliquots were transferred to other tubes.The Pi released was measured according to the method of Fiskeand Subbarow (Fiske and Subbarow, 1925). Briefly, the releasedphosphate was added to a solution containing 8% ferrous sulfate(w/v), 50% ammonium molybdate (v/v) and 20% sulfuric acid (v/v) to produce a complex of ammonium phosphomolybdate. Thissolution was measured spectrophotometrically at 650 nm, and aPi curve was used as a standard. In experiments that tested highconcentrations of MnCl2 and CaCl2, the possibility of precipitateformation was checked as described previously (Meyer-Fernandesand Vieyra, 1988). In reaction media containing 50 mM HEPES (pH7.4), 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose and 1 mM PPi,no phosphate precipitates were observed in the presence of thesecations under the conditions used.

2.4. Statistical analysis

All experiments were performed in triplicate, and similar re-sults were obtained from at least three separate cell suspensions.The data were analyzed statistically by Student’s t-test or a one-way ANOVA followed by the Tukey test using Prism computer soft-ware (Graphpad Software Inc., San Diego, CA, USA). A result wasconsidered to be statistically significant when p < 0.05.

3. Results

In this study, we identified and characterized an ecto-pyrophos-phatase activity found at the external surface of the plasma mem-brane of L. amazonensis promastigotes. Because the presence ofbroken cells or the breakage of cells under the assay led to the

Fig. 1. Time course and cell density dependence of the ecto-PPase activity of L. amazonensis. Intact procyclic promastigote forms of L. amazonensis were incubated at roomtemperature in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 50 mM HEPES–Tris (pH 7.2), 1 mM PPi and 5 mM MgCl2 for 10–60 min (A).Increasing concentrations of cells were incubated for 1 h at room temperature in the same reaction medium described above (B).

Fig. 2. Effect of MnCl2 or MgCl2 on the ecto-PPase activity of L. amazonensis. Intactprocyclic promastigote forms of L. amazonensis were incubated for 1 h at roomtemperature in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mMD-glucose, 50 mM HEPES–Tris (pH 7.2), 1 mM PPi and 1 mM MnCl2 or MgCl2. Theasterisk denotes significant stimulation compared with the basal activity (withoutthe addition of any metal).

Fig. 3. Effect of increasing concentrations of MgCl2 on ecto-pyrophosphatase, ecto-b-glycerophosphatase and ecto-phosphatase activities of L. amazonensis. Intactprocyclic promastigote forms of L. amazonensis were incubated for 1 h at roomtemperature in a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mMD-glucose, 50 mM HEPES–Tris (pH 7.2), 1 mM of substrate (PPi, b-GP or p-NPP) andincreasing concentrations of MgCl2. The graph shows: ecto-PPase activity (d); ecto-b-glycerophosphatase activity (s); and ecto-phosphatase activity (N).

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hydrolysis of substrates and produced false positive data (Furuyaet al., 1998; Dick et al., 2010), we first determined whether therewas any contribution from intracellular PPase activity. Therefore,cellular viability was assessed before and after incubations bymotility and Trypan blue dye exclusion. Under the conditions em-ployed in all of the experiments, the cell viability was not signifi-cantly affected. Moreover, to ensure that the PPi hydrolysispromoted by living promastigotes did not result from secreted en-zymes, cells were incubated in a reaction mixture containing noPPi. The cells were removed by centrifugation, and the supernatantwas assayed for pyrophosphatase activity. This supernatant failedto show evidence of PPi hydrolysis (data not shown). These dataexclude the possibility that the PPi hydrolysis found in living cellscould be derived from secreted enzymes or lysed L. amazonensispromastigotes.

The time course of the ecto-PPase activity of L. amazonensis waslinear for at least 1 h (Fig. 1A). Similarly, in an assay designed todetermine the influence of cell density on ecto-PPase activity, weobserved that this activity was directly proportional to the numberof cells (Fig. 1B). Therefore, the following experiments were per-formed with 2 � 107 cells per mL over 1 h. Under these conditions,PPi was hydrolyzed at a rate of 46.52 ± 4.10 nmol Pi � h�1 � 10�7

cells.The catalytic activity of most described pyrophosphatases is

stimulated by divalent metals, especially magnesium and manga-nese. To determine whether these metals could modulate theecto-PPase activity of L. amazonensis, hydrolysis of PPi was mea-sured in the absence of these metals or in the presence of 1 mMMgCl2 or MnCl2. The ecto-PPase activity measured in the absenceof divalent metals was significantly lower than the ecto-PPaseactivity measured in the presence of MgCl2. However, MnCl2 didnot increase the pyrophosphate hydrolysis (Fig. 2).

As shown in Fig. 3, MgCl2 stimulated the hydrolysis of PPi in adose-dependent manner and had an apparent K0.5 value of1.59 ± 0.18 mM MgCl2. However, MgCl2 did not stimulate thehydrolysis of two other phosphorylated substrates, p-nitrophenylphosphate (p-NPP) and b-glycerophosphate (b-GP). These dataindicate that the hydrolysis of PPi, p-NPP and b-GP is catalyzedby different ecto-enzymes.

Another biochemical parameter of the ecto-PPase activity of L.amazonensis analyzed was the response to pH variation. We testedthe parasites in the pH range from 5.8 to 8.4, in which the parasiteswere viable during the entire course of the reaction. We found thatthe ecto-PPase activity increased in the alkaline pH range (Fig. 4A).Even at an alkaline pH (8.4), levamizole, an alkaline phosphataseinhibitor, could not modulate the ecto-PPase activity. In contrast,a progressive reduction of the ecto-phosphatase activity was ob-served over the same pH range (Fig. 4B). These results further sug-gest that the hydrolysis of extracellular PPi in L. amazonensis is notrelated to phosphatase activity.

In addition to levamizole, other inhibitors were tested for ef-fects on the ecto-PPase activity at a neutral pH (7.2). As shown inTable 1, sodium orthovanadate, an inhibitor of acid phosphatases,and tartrate, an inhibitor of secreted phosphatases, did not signif-icantly modulate ecto-PPase activity. However, sodium fluoride(NaF), an inhibitor of pyrophosphatases, almost completelyabolished the ecto-PPase activity at a concentration of 1 mM. Theinhibition of the ecto-PPase activity by NaF was dose-dependentand had an apparent Ki value of 0.34 ± 0.03 lM NaF (Fig. 5A). Cal-cium also inhibited the ecto-PPase activity (Fig. 5B). Increasingconcentrations of this metal inhibited Mg2+-stimulated activity

Fig. 4. Effect of pH on ecto-PPase and ecto-phosphatase activities of L. amazonensis. Intact procyclic promastigote forms of L. amazonensis were incubated for 1 h in a reactionmedium containing 5 mM MgCl2 and 1 mM PPi (A) or 1 mM p-NPP (B), and a buffer solution with 100 mM citrate/Mes/Tris adjusted to pH values from 5.8 to 8.4. In panel A,the symbol (s) represents the effect of 10 mM levamizole on the ecto-PPase activity at alkaline pH (8.4).

Fig. 6. Ecto-PPase activity during L. amazonensis proliferation. Intact procyclicpromastigote forms of L. amazonensis were incubated for 1 h at room temperaturein a reaction medium containing 116 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose,50 mM HEPES–Tris (pH 7.2), 1 mM PPi and 5 mM MgCl2. The cells used were grownfor 1 to 7 days. The graph shows the ecto-PPase activity (s) and the number of cellspresent in the culture medium on each of the days (d).

Table 1Effect of several inhibitors on the ecto-PPase activity of L. amazonensis.

Intact procyclic promastigote forms of L. amazonensis were incubated for 1 h atroom temperature in a reaction medium containing 116 mM NaCl, 5.4 mM KCl,5.5 mM D-glucose, 50 mM HEPES–Tris (pH 7.2), 1 mM PPi, 5 mM MgCl2 and 1 mM ofthe indicated inhibitor. The asterisk denotes significant inhibition compared withthe enzyme activity of the control (no inhibitor).

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with an apparent Ki value of 2.34 mM CaCl2 and had no effect onecto-PPase activity in the absence of MgCl2. These data indicatethat the inhibition exerted by CaCl2 may be due to competition be-tween Ca2+ and Mg2+.

After analyzing these biochemical parameters, the profile of theecto-PPase activity over several days of cell culture was deter-mined. As shown in Fig. 6, the ecto-PPase activity decreased withthe growth phase of the parasite, with 2.5-fold higher ecto-PPaseactivity on the first day than on the seventh day.

4. Discussion

Little is known about extracellular PPi hydrolysis, especially inprotozoan parasites. Recently, a membrane-bound pyrophospha-tase was described in the plasma membrane of L. donovani.Although most of the activity is located in the inner membrane,an ecto-PPase activity was observed at the exoplasmic face of the

Fig. 5. Effect of increasing concentrations of NaF and CaCl2 on the ecto-PPase activity of Lfor 1 h at room temperature in a reaction medium containing 116 mM NaCl, 5.4 mMincreasing concentrations of NaF (A) or CaCl2 (B). Panel B shows the inhibition of the Mg2+

the addition of MgCl2) (s).

plasma membrane (Sen et al., 2009). In this study, an ecto-PPaseactivity was identified and characterized in promastigote formsof L. amazonensis.

Although inorganic pyrophosphatases are divided into two dis-tinct groups, the soluble PPases and the H+-PPases both presentsimilar biochemical profiles. The activity of most of the pyrophos-phatases is stimulated by or dependent on Mg2+ (Cooperman et al.,1992; Leigh et al., 1992). However, the members of family II sPPas-es have a preference for Mn2+ instead of Mg2+ (Zyryanov et al.,2004). The ecto-PPase activity from L. amazonensis was stimulatedby MgCl2 but did not show any activity in the presence of the sameconcentration of MnCl2. This result indicates that the ecto-PPase of

. amazonensis. Intact procyclic promastigote forms of L. amazonensis were incubatedKCl, 5.5 mM D-glucose, 50 mM HEPES–Tris (pH 7.2), 1 mM PPi, 5 mM MgCl2 and-dependent ecto-PPase activity by CaCl2 (d) and the effect on basal activity (without

12 A.L. Freitas-Mesquita et al. / Experimental Parasitology 137 (2014) 8–13

L. amazonensis and the members of family I sPPases have a similarprofile.

Several ecto-enzymatic activities present in the plasma mem-brane of L. amazonensis have already been characterized by ourgroup (Berrêdo-Pinho et al., 2001; Pinheiro et al., 2006; De Almei-da-Amaral et al., 2006; Paletta-Silva et al., 2011). Among them, theecto-phosphatase activity is involved in hydrolyzing phosphory-lated substrates to make inorganic phosphate available for the par-asite (De Almeida-Amaral et al., 2006). Because pyrophosphate is aphosphorylated substrate, we performed experiments to show thatthe enzyme is a genuine pyrophosphatase rather than a phospha-tase. First, we verified that the hydrolysis of PPi was stimulatedin a dose-dependent manner by MgCl2. In contrast, the hydrolysisof p-NPP and b-GP, two substrates of the ecto-phosphatase, are notaffected by MgCl2. Furthermore, the ecto-PPase activity was stim-ulated at alkaline pH range while the ecto-phosphatase activitywas inhibited at the same pH range. Finally, while 1 mM sodiumorthovanadate almost abolished the ecto-phosphatase activity(De Almeida-Amaral et al., 2006), no inhibition was observed inthe hydrolysis of PPi.

Because the ecto-PPase activity from L. amazonensis has an alka-line profile, it was important to exclude the possibility of participa-tion of a nonspecific alkaline phosphatase. Levamizole, a classicalalkaline phosphatase inhibitor, was tested and did not inhibit theecto-PPase activity, even at alkaline pH. Moreover, this activitycannot be attributed to a secreted phosphatase because the super-natant from the cells failed to show PPi hydrolysis, and the ecto-PPase was not inhibited by tartrate, which is a classical secretedphosphatase inhibitor (Fernandes et al., 2013). Because of the dif-ferent enzyme activity profiles in response to the tested inhibitors,we can exclude the participation of ecto-phosphatases or secretedphosphatases in PPi hydrolysis. This approach has been used by ourgroup for other enzymatic characterizations such as that of theecto-ATPase activity in Leishmania tropica (Meyer-Fernandeset al., 1997) and the ecto-phosphatase activities of L. amazonensisDe Almeida-Amaral et al., 2006; Fernandes et al., 2013).

Among the classical phosphatase inhibitors that were tested forthe inhibition of ecto-PPase activity, only sodium fluoride (NaF)was capable of inhibiting this hydrolysis. Although fluoride is aninhibitor of several types of enzymes, pyrophosphatase is the mostsensitive (Baykov et al., 1992). The ecto-PPase activity of L. ama-zonensis is sensitive to NaF, reaching maximal inhibition (ofapproximately 80%) in the presence of 20 lM of the inhibitor. Thisconcentration range is consistent with the result described by Bay-kov et al. (1992) for rat liver inorganic pyrophosphatase.

Another effective inhibitor of ecto-PPase from L. amazonensis isCaCl2. A very detailed mechanism of inhibition by Ca2+ during PPihydrolysis was described for the inorganic PPase from Escherichiacoli, for which the mechanism of PPi hydrolysis is well established(Rodina et al., 2001). Because PPases from various organisms exhi-bit similarity in active site structure, these enzymes likely share acommon mechanism of catalysis (Avaeva et al., 2000). PPase activ-ity requires at least three Mg2+ ions. In the active site of the en-zyme, four metal-binding sites, designated M1–M4, have beenidentified. In the absence of a substrate, the first two Mg2+ bindto the M1 and M2 sites. The third Mg2+ interacts with PPi to formthe PPase substrate, which consists of the Mg2+PPi complex. Whenthere is an abundance of Mg2+, a fourth Mg2+ binds in the activesite and inhibits PPase activity. Because the inhibition does not ap-pear to be competitive, it is hypothesized that the Mg2+PPi com-plex binds to the M3 site and the fourth Mg2+ binds to the M4site, although the mechanism has not yet been clearly defined(Rodina et al., 2001). Once the M2 site has a higher affinity forCa2+, this metal can easily replace the Mg2+ bound in this regionof the active site. However, the binding of Ca2+ cannot activatethe enzyme. Furthermore, Ca2+ can compete with Mg2+ to form a

complex with PPi; however, the Ca2+PPi complex is not a substratefor the enzyme. Because of the similarity of the active sites of theenzymes, these mechanisms of inhibition can be extrapolated toother organisms (Avaeva et al., 2000).

The ecto-PPase activity from L. amazonensis was determined onvarious days of parasite growth and was found to be significantlyhigher during the first days of culture. In this period, which is des-ignated as the exponential phase, the parasites are constantly pro-liferating. Interestingly, higher ecto-phosphatase activity was alsoobserved for Trypanosoma rangeli during the first days of growth(Fonseca-de-Souza et al., 2009). These results lead us to hypothe-size that these parasites activate ecto-enzymatic activities relatedto phosphate metabolism during the exponential phase of growth.

In this study, we characterized, for the first time, an ecto-PPaseactivity of L. amazonensis promastigotes. This activity shares manyfeatures with other well-known inorganic pyrophosphatases, suchas stimulation by MgCl2 and inhibition by CaCl2 and NaF. The char-acterization of another ecto-enzyme involved in phosphate acqui-sition from protozoan parasites is important to furtherunderstanding the mechanisms of phosphate metabolism. More-over, pyrophosphate was recently associated with the inflamma-tion processes, which opens new possibilities for studying thepossible roles of this molecule in Leishmania infection (Lopez-Cast-ejón et al., 2011). Further studies are required to completely eluci-date the role of this ecto-enzyme in the development of theseparasites.

Acknowledgments

We would like to thank Mr. Fabiano Ferreira Esteves and Ms.Rosangela Rosa de Araújo for their excellent technical assistance.This work was supported by Grants from the Brazilian AgenciesConselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES) and Fundação de Amparo à Pesquisa do Estadodo Rio de Janeiro (FAPERJ).

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