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CrATP as a new inhibitor of ecto-ATPases of trypanosomatids

Published online by Cambridge University Press:  07 January 2009

O. C. MOREIRA ,
P. F. RIOS ,
F. F. ESTEVES ,
J. R. MEYER-FERNANDES  and
H. BARRABIN
O. C. MOREIRA
Affiliation:
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro de Ciências da Saúde, Universidade Federal do Rio de JaneiroRio de Janeiro, Brasil
P. F. RIOS
Affiliation:
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro de Ciências da Saúde, Universidade Federal do Rio de JaneiroRio de Janeiro, Brasil
F. F. ESTEVES
Affiliation:
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro de Ciências da Saúde, Universidade Federal do Rio de JaneiroRio de Janeiro, Brasil
J. R. MEYER-FERNANDES
Affiliation:
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro de Ciências da Saúde, Universidade Federal do Rio de JaneiroRio de Janeiro, Brasil
H. BARRABIN*
Affiliation:
Instituto de Bioquímica Médica, Programa de Biologia Estrutural, Centro de Ciências da Saúde, Universidade Federal do Rio de JaneiroRio de Janeiro, Brasil
*
*Corresponding author: Instituto de Bioquímica Médica, CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, CEP 21941-590, Rio de Janeiro, RJ, Brazil. Fax: +55 21 2270 8647. E-mail: barrabin@bioqmed.ufrj.br
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Summary

Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5′-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4·7±1·0 μm but a fraction of about 40–50% was insensitive to CrATP. Remarkably, at low substrate concentration (0·2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0·33±0·10 μm. These parameter dependences are interpreted as the presence of 2 ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2′ disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of ecto-ATPases and as a tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites.

Keywords

ecto-ATPaseHerpetomonasCrATPtrypanosomatidsecto-phosphatase
Type
Research Article
Information
Parasitology , Volume 136 , Issue 1 , January 2009 , pp. 35 - 44
Copyright
Copyright © 2009 Cambridge University Press

INTRODUCTION

Trypanosomatid protozoa include heteroxenic species that are pathogenic for men (e.g. disease-causing parasites such as Leishmania and Trypanosoma), pathogenic for plants (genus Phytomonas and some Herpetomonas spp.) and non-pathogenic species that develop strictly in the insect midgut (e.g. Crithidia).

Over 20 different species of the genus Leishmania are known to be pathogenic for humans. It is estimated that more than 2 million new cases of leishmaniases arise each year (Desjeux, Reference Desjeux2004). Chagas disease is widespread throughout Latin America, where nearly 20 million people are infected by Trypanosoma cruzi and 90 million are at risk in endemic areas (World Health Organization, 2002).

Infection of plants with trypanosomatids has been known since 1909, when Lafont (Reference Lafont1909) described these flagellates in laticiferous plants. The genus Phytomonas and some Herpetomonas parasitize plants without apparent pathogenicity but they can also cause diseases of economic significance in plantations of coconut, oil palm, cassava and coffee (Dollet, Reference Dollet1984; Camargo et al. Reference Camargo, Kastelein and Roitman1990; Redman et al. Reference Redman, Schineider, Mehlert and Ferguson1995; Camargo, Reference Camargo1999; Catarino et al. Reference Catarino, Serrano, Covazzana, Almeida, Kaneshima, Campaner, Jankevicius, Teixeira and Itow-Jankevicius2001). These trypanosomatids have been detected in various edible fruits, such as guavas, peaches, pomegranates and tangerines and in their insect vectors (Redman et al. Reference Redman, Schineider, Mehlert and Ferguson1995). The parasites live mostly in the xylem and phloem of the infected plants and are transmitted through the bite of phytophagous insects (Dollet, Reference Dollet1984; Camargo et al. Reference Camargo, Kastelein and Roitman1990; Redman et al. Reference Redman, Schineider, Mehlert and Ferguson1995; Camargo, Reference Camargo1999; Catarino et al. Reference Catarino, Serrano, Covazzana, Almeida, Kaneshima, Campaner, Jankevicius, Teixeira and Itow-Jankevicius2001). In the biological cycle of these pathogens, several plant-sucking insects act as intermediate hosts and the plant acts as the main host (Jankevicius et al. Reference Jankevicius, Jankevicius, Campaner, Conchon, Maeda, Teixeira, Freymuller and Camargo1989). The promastigotes form predominates in both axenic cultures and in isolates from plants and insects and shows ultrastructural features typical of the family Trypanosomatidae, containing kinetoplast, glycosomes, endoplasmic reticulum, acidocalcisomes and a single tubular mitochondrion (Attias et al. Reference Attias, Roitman, Camargo, Dollet and De Souza1988; Freymuller et al. Reference Freymuller, Milder, Jankevicius, Jankevicius and Camargo1990; Redman et al. Reference Redman, Schineider, Mehlert and Ferguson1995; Soares de Medeiros et al. Reference Soares de Medeiros, Moreira, Kildare, de Souza, Plattner, Hentschel and Barrabin2005).

Surface-mediated interactions between any of these parasites and the media environment are of critical importance from the physiological point of view. Parasite membrane contains 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 intact cells (De Pierre and Karnowsky, Reference De Pierre and Karnovsky1973, Reference De Pierre and Karnovsky1974; Furuya et al. Reference Furuya, Zhong, Meyer-Fernandes, Lu, Moreno and Docampo1998; Meyer-Fernandes, Reference Meyer-Fernandes2002).

The ecto-NTPases (EC 3.6.1) were referred to usually as ecto-nucleoside triphosphate diphosphohydrolases 2 (ecto-NTDPase 2) type or ecto-ATPases (E-type ATPases) since they hydrolyse ATP better than other trinucleotides while ADP is hardly hydrolysed. Cell membrane ecto-ATPases are integral membrane glycoproteins, millimolar divalent cation-dependent, that hydrolyse all nucleoside triphosphates (Plesner, 1995; Kirley, Reference Kirley1997; Meyer-Fernandes et al. Reference Meyer-Fernandes, Dutra, Rodrigues, Saad- Nehme and Lopes1997). The ecto-apyrases (EC 3.6.1.5) or NTDPases 1, on the other hand, can hydrolyse ADP to AMP+Pi as efficiently as ATP to ADP+Pi. Although both enzymes exhibit a slightly different sensitivity to inhibitors (Plesner, Reference Plesner1995; Heine et al. Reference Heine, Braun, Heilbronn and Zimmermann1999), they share close sequence homology (Handa and Guidotti, Reference Handa and Guidotti1996). For a review about the identity and the function of mammalian ecto-NTDPases see Robson et al. (Reference Robson, Sévigay and Zimmermann2006). Both enzymes are unaffected by the ecto-phosphatase inhibitors (Meyer-Fernandes, Reference Meyer-Fernandes2002). It has been reported that the ecto-ATPases are sensitive to dicyclohexylcarbodiimide (DCCD) and suramin, which are compounds that do not cross the cellular membrane.

Ecto-ATPases have been described in several protozoa such as Toxoplasma gondii (Asai and Suzuki, Reference Asai and Suzuki1990; Bermudes et al. Reference Bermudes, Peck, Afifi, Beckers and Joiner1994; Asai et al. Reference Asai, Miura, Sibley, Okabayashi and Takeuchi1995; Nakaar et al. Reference Nakaar, Beckers, Polotsky and Joiner1998), Entamoeba histolytica (Bakker-Grunwald and Parduhn, Reference Bakker-Grunwald and Parduhn1993; Barros et al. Reference Barros, De Menezes, Pinheiro, Silva, Lopes, De Souza and Meyer-Fernandes2000), Tetrahymena thermophila (Smith et al. Reference Smith, Kirley and Hennessey1997), Leishmania tropica (Meyer-Fernandes et al. Reference Meyer-Fernandes, Dutra, Rodrigues, Saad- Nehme and Lopes1997), L. amazonensis (Berredo-Pinho et al. Reference Berredo-Pinho, Peres-Sampaio, Chrispim, Belmont-Firpo, Lemos, Martiny, Vannier-Santos and Meyer-Fernandes2001), Trypanosoma cruzi (Bernardes et al. 2001; Bisaggio et al. Reference Bisaggio, Peres-Sampaio, Meyer-Fernandes and Souto-Padron2003; Meyer-Fernandes et al. Reference Meyer-Fernandes, Saad-Nehme, Peres-Sampaio, Belmont-Firpo, Bisaggio, Do Couto, Fonseca De Souza, Lopes and Souto-Padron2004), Herpetomonas muscarum (Alves-Ferreira et al. Reference Alves-Ferreira, Dutra, Lopes, Ferreira-Pereira, Scofano and Meyer-Fernandes2003), Tritrichomonas foetus (de Jesus et al. Reference de Jesus, de Sa Pinheiro, Lopes and Meyer-Fernandes2002), Crithidia deanei (dos Passo Lemos et al. Reference dos Passos Lemos, de Sa Pinheiro, de Berredo-Pinho, Fonseca de Souza, Motta, de Souza and Meyer-Fernandes2002).

In mammals, the ecto-ATPases seem to have multiple roles in extracellular nucleotide metabolism and in the regulation of nucleotide-based intercellular signalling (Plesner, Reference Plesner1995; Robson et al. Reference Robson, Sévigay and Zimmermann2006). However, their physiological roles in protozoa parasites are poorly understood. Experimental evidence suggests their involvement in the adenosine acquisition mechanisms and in the mechanism of parasite infection (for a review see Meyer-Fernandes, Reference Meyer-Fernandes2002).

The bidentate complex of ATP with Cr3+, CrATP, is an MgATP analogue that is known to form a stable complex with P-ATPases provoking their inhibition. This mechanism has been described for Na+,K+-ATPase (Pauls et al. Reference Pauls, Bredenbröcker and Schoner1980; Linnertz et al. Reference Linnertz, Thönges and Schoner1995) sarcoplasmic reticulum Ca2+-ATPase (Vilsen and Andersen, Reference Vilsen and Andersen1992; Vilsen, Reference Vilsen1995; Einholm et al. Reference Einholm, Vilsen and Andersen2004) and the PMCA (Moreira et al. Reference Moreira, Rios and Barrabin2005). CrATP has also been shown to inhibit other enzymes that have ATP as a substrate, like phosphoglycerate kinase (Serpersu et al. Reference Serpersu, Summitt and Gregory1992), fructokinase (Raushel and Cleland, Reference Raushel and Cleland1977), ADP-glucose pyrophosphorylase (Binderup et al. Reference Binderup, Watanabe, Polikarpov, Preiss and Arni2000) and hexokinase (Danenberg and Cleland, 1975).

The molecular diversity and cellular functions of ecto-NTDPases in protozoa parasites are still an open question (Zimmermann, Reference Zimmermann1999). In this regard, the development of specific enzyme inhibitors promises to offer important tools for basic research and therapy. Here we show that CrATP is an efficient inhibitor of the ecto-ATPase from Herpetomonas sp. and other pathogenic trypanosomatids, but does not inhibit either the Mg-independent ADPase or the p-nitrophenylphosphatase activities. Data suggest that the ecto-ATPase in Herpetomonas is not a single enzyme but the mixture of 2 different isozymes, only one of them being sensitive to CrATP.

MATERIALS AND METHODS

Reagents

All reagents were purchased from E. Merck (Darmstadt, Germany) or Sigma Chemical Co. (St Louis, MO). [γ-32P]ATP was synthesized as described by Walseth and Johnson (Reference Walseth and Johnson1979).

CrATP synthesis

CrATP was synthesized from the sodium salt of ATP and CrCl3, as described by De Phamphilis and Cleland (1973). The bidentate isomer was isolated through a cation-exchange column (Dowex 50-H+ resin) and the product confirmed by the absorption spectrum between 400 and 800 nm (De Phamphilis and Cleland, 1973) and quantified by its absorption at 610 nm (ε610 nm=20 M−1. cm−1). The final product is free of contaminating Cr3+ since addition of 1 mm citrate (an efficient Cr3+ chelator) to the reaction medium does not modify the inhibition by CrATP (not shown).

Culture methods

Herpetomonas sp. promastigotes (CT.IOC 13; kindly provided by Dr Reginaldo Brazil, Instituto Rene Rachour, FIOCRUZ, MG, Brazil) were grown at 25°C in a medium containing 20 g/l sucrose, 20 g/l KCl, 3 g/l yeast extract, 3 g/l peptone, 1 mg/l folic acid, supplemented with 10 mg/l haemin and 10% (v/v) fetal bovine serum. At 2 days after inoculation, cells were harvested by centrifugation, washed twice with 5 mm phosphate buffer, pH 7·0, containing 150 mm NaCl (PBS) and resuspended in the same solution. Trypanosoma rangeli (Macias strain, kindly supplied by Dr Wanderley De Souza, UFRJ, Brasil) was used throughout this study. The parasites were grown in liver infusion tryptose medium supplemented with 20% fetal calf serum at 28°C. Five days after inoculation, long epimastigotes, predominantly (>98%), were collected by centrifugation, washed twice and kept in 50 mm Tris-HCl buffer, pH 7·2, 100 mm sucrose and 20 mm KCl and resuspended in the same solution. Trypanosoma cruzi (Y strain) were grown in liver infusion tryptose medium supplemented with 10% fetal calf serum at 28°C. Seven days after inoculation, the parasites were harvested by centrifugation, washed twice with 0·9% saline and once with 30 mm Tris-HCl/75 mm sucrose buffer, pH 6·8, and kept in the same buffer before the assays. The MHOM/BR/75/Josefa strain of Leishmania amazonensis was used throughout this study. It was isolated from a human case of diffuse cutaneous leishmaniasis in Brasil and provided by Dr Cuba-Cuba (Universidade de Brasilia, Distrito Federal, Brasil) and has been maintained in our laboratory in axênica culture. Promastigotes were cultured in Warren′s medium supplemented with 10% heat-inactivated fetal calf serum at 22°C. Five days after inoculation, promastigotes were collected by centrifugation, washed twice and kept in 116 mm NaCl, 5·4 mm KCl, 5·5 mm D-glucose, and 50 mm Hepes-Tris (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid adjusted with hydroxymethyl aminomethane) buffer, pH 7·2, and kept in the same buffer before the assays. For all parasites the cell number was measured in a Neubauer chamber. Cellular viability was assessed before and after incubation using motility and trypan blue dye exclusion assessments.

Ecto-ATPase activity

All intact parasites were incubated for 1 h at 30°C in 500 μl of a mixture (standard medium) containing, unless otherwise specified, 116 mm NaCl, 5·4 mm KCl, 5·5 mm D-glucose, 50 mm Hepes-Tris buffer (pH 7·2), 2 mm ATP and 1·0×108 cells/ml in the absence or presence of 2 mm MgCl2. 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. ATPase activity was determined by measuring the hydrolysis of [γ-32P]ATP (104 Bq/nmol ATP).

The experiments were started by the addition of living cells and stopped by the addition of 1 ml of a cold mixture containing 200 mg charcoal in 1 m HCl. The tubes were centrifuged at 1500 g for 10 min at 4°C. Aliquots (500 μl) of the supernatants containing the released inorganic phosphate (32Pi) were deposited on filtering paper and, after drying, transferred to scintillation vials containing 9 ml of scintillation fluid (2 g 2,5-diphenyloxazole (PPO) in 1 l of toluene). ATPase activity was calculated by subtracting the non-specific ATP hydrolysis measured in the absence of cells. ATP hydrolysis was linear with time under the assay conditions used and was proportional to cell numbers for all parasites.

Alternatively, using ADP or ATP as substrates, the hydrolytic activities, under the same conditions as described above, were assayed spectrophotometrically by measuring the release of Pi from the nucleotides (Lowry and Lopes, Reference Lowry and Lopes1946). The values obtained for ATPase activities measured using both methods (spectrophotometric and radioactive) were exactly the same.

E-Phosphatase activity

In addition to the measurements of ecto-ATPase activity, ecto-para-nitrophenylphosphatase activity was determined in the same medium as that for ATP hydrolysis, except that ATP was replaced by 5 mm p-nitrophenylphosphate. The reaction was determined spectrophotometrically at 425 nm, using a molar extinction coeficient of ε =14·3×103 M−1.cm−1.

RESULTS

As described for other trypanosomatids, Herpetomonas sp. presented a Mg2+-dependent ecto-ATPase activity on its external surface (Fig. 1). In the intact parasites, whose viability was assessed before and after the reactions by motility and by trypan blue dye exclusion, ATP hydrolysis was low (4·5±0·9 nmol Pi/h/108 cells) in the absence of any divalent metal (addition of 1 mm ethylenediaminetetraacetic acid (EDTA)). At pH 7·2, the addition of 2 mm MgCl2 stimulated the ATP hydrolysis up to 80·2±3·0 nmol Pi/h/108 cells, resulting in a Mg2+-dependent ecto-ATPase activity of 75·7±3·1 nmol Pi/h/108 cells. The time-course of ATP hydrolysis by the Herpetomonas sp. ecto-ATPase was linear for at least 60 min (Fig. 1A). Similarly, in assays to determine the influence of cell density, Pi formation was linear over a nearly 10-fold range of cell density (Fig. 1B). To check the possibility that the observed ATP hydrolysis was the result of secreted soluble enzymes, as seen in other parasites, 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). These data also rule out the possibility that the ATPase activity here described could be from lysed Herpetomonas sp. cells.

Fig. 1. Time-course (A) and effect of cell density (B) on the ecto-ATPase activity by intact cells of Herpetomonas sp. Cells were incubated for different periods of time (A) or for a fixed period of 1 h (B) at 37°C. The reaction medium contained 50 mm Tris–HCl buffer, pH 7·2, 2 mm Tris–ATP [γ-32P]ATP (sp. act 104 Bq/nmol ATP), 2 mm MgCl2 and 1 mg/ml of protein which corresponds to 2·2×108 cells/ml. Data are means±s.d. of 3 determinations with different cell suspensions.

Addition of CrATP to the hydrolytic media partially inhibited the Mg2+-dependent ecto-ATPase activity with a Ki of 4·8±1·0 μm (Fig. 2A). The inhibition was not affected by pre-incubation of the cells for 1 h with CrATP (not shown). Furthermore, washing the cells after 1 h exposure with 200 μm CrATP fully restored the ecto-ATPase activity, indicating that the inhibition was reversible. The Mg2+-independent ecto-ATPase activity was insensitive to CrATP (Fig. 2A). Tests performed with 1 mm CrATP, in the absence of other nucleotides, showed that this analogue was not hydrolysed by the ecto-enzymes of Herpetomonas sp. (not shown).

Fig. 2. Effect of increasing concentrations of CrATP on the ecto-ATPase (A) and phosphatase (B) activities of Herpetomonas sp. (A) Intact cells were incubated for 1 h at room temperature, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm [γ-32P]ATP (4000 cpm/nmol), in the absence (○) or presence (•) of 2 mm MgCl2. In the case of absence of MgCl2, 300 μm EDTA was added. Data are means±s.e. of 3 determinations with different cell suspensions. The black square shows the reversibility of ecto-ATPase inhibition. Cells were incubated at room temperature with 200 μm CrATP as above and washed by centrifugation. Ecto-ATPase activity was assayed with 2 mm [γ-32P]ATP. Values representing 100% activities were 4·90±0·04 (○) and 79·5±15 (•) nmol Pi/108 cel×h, respectively. (B) Intact cells were incubated for 1 h, at room temperature, in medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and increasing concentrations of CrATP, in the presence of 5 mmp-nitrophenylphosphate (pNPP). Data are means±s.e. of 3 determinations with different cell suspensions. 100% activity was 94±10 nmol pNP/108 cel×h.

Several ecto-enzymes were described in trypanosomatids that can hydrolyse phosphorylated compounds: ecto-ATPase (NTPDase1), ecto-nucleoside triphosphate diphosphohydrolases (NTPDase2) and ecto-phosphatase. To check if these enzymes were affected by CrATP different substrates were used. The ecto-phosphatase activity, followed by pNPP hydrolysis, was only slightly inhibited (10%) by the ATP analogue (Fig. 2B). The NTPDases2 are enzymes that can hydrolyse ATP to ADP as well as ADP to AMP. The Herpetomonas sp. cells were able to hydrolyse ADP at low levels; however, this activity was not stimulated by Mg2+ so it must not be a truly NTPDase2. Futhermore, the CrATP did not inhibit the Mg2+ independent ADP hydrolysis at concentrations able to cause maximal inhibition of ecto-ATPase (results not shown).

The curve of inhibition of ecto-ATPase at a 10-fold lower ATP concentration (0·2 mm) gives a Ki approximately 10 times lower (0·33±0·04 μm) suggesting a competitive mechanism (Fig. 3A). Curiously, the degree of maximal inhibition increased with the decrease of substrate concentration. This effect is better described in Fig. 3B, where the full ecto-ATPase activities, the maximally inhibited (residual) and the CrATP sensitive activities measured at different ATP concentrations were plotted. With the increase in the ATP concentration appeared a CrATP-resistant ecto-ATPase activity that seemed to have Km=3·49±0·19 mm and Vmax=115·6±3·2 nmol Pi/108 cel×h. The CrATP sensitive activity seemed to have a much higher apparent affinity for ATP (Km=85±39 μm) and a lower maximal velocity than the insensitive one (Vmax=39·2±3·9 nmol Pi/108 cel×h).

Fig. 3. ATP dependence of the CrATP inhibition of the ecto-ATPase activity. (A) Intact cells were incubated for 1 h at room temperature, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and increasing concentrations of CrATP, in the presence of (▪) 200 μm [γ-32P]ATP (4000 cpm/nmol) or (•) 2 mm [γ-32P]ATP (400 cpm/nmol). Data are means±s.e. of 3 determinations with different cell suspensions. Data were fitted by non-linear regression using the equation v=100−As/(1+Ki/[CrATP]), where v is enzyme activity, As represents the sensitive maximal activity (in %) and Ki the concentration of CrATP to inhibit half of the As. The activity at ‘infinite’ CrATP concentration, Ai=100−As, represents the percentage of insensitive activity. In (▪) 100% activity was 32·1±1·4 nmol Pi/108 cel×h cells and Ki=0·33±0·10 μm; in (•) 100% activity was 79·5±15 nmol Pi/108 cel×h and Ki=4·7±1·0 μm. (B) Activities measured in the absence (○) and in the presence (•) of 200 μm CrATP at different ATP concentrations. The difference between the curves (CrATP sensitive ecto-ATPase) was calculated (▴). The lines represent fitting of the data with the Michaelis-Menten equation (▴,○). The continuous line, representing the fit of the total activity, was calculated as the sum of the fitted sensitive and insensitive activities.

DIDS and suramin are impermeable compounds that had been used as inhibitors of ecto-ATPases of trypanosomatids (Bisaggio et al. Reference Bisaggio, Peres-Sampaio, Meyer-Fernandes and Souto-Padron2003). The ecto-ATPase activity of Herpetomonas sp. was effectively inhibited by these two polyanions (Fig. 4A and B). The apparent Ki's were 19·0±5·1 and 15·2±7·6 μm respectively. It should be noted that inhibition by DIDS was also partial and higher at 0·2 mm (82%) than at 2 mm ATP (42%).

Fig. 4. Effect of DIDS (A) and suramin (B) on ecto-ATPase activity of Herpetomonas sp. Intact cells were incubated for 1 h at room temperature, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and increasing concentrations of (A) DIDS in the presence of (○) 200 μm or (•) 2 mm [γ-32P]ATP or (B) suramin in the presence of 2 mm [γ-32P]ATP. Data are means±s.e. of 3 determinations with different cell suspensions.

Bearing in mind that the compounds used here have partial inhibitory effects and taking into consideration the possibility of having more than one enzyme with different inhibitor sensitivities, the maximal inhibitory effects of associations of CrATP+DIDS or CrATP+suramin were measured (Fig. 5). Additive effects of inhibitors were observed, suggesting that they act on different iso-enzymes or that they act on different regions of one enzyme, each one having a partial and independent inhibitory effect. The dependence of inhibition on ATP concentration seems to argue against this last idea.

Fig. 5. Additive effects of DIDS or suramin and CrATP on ecto-ATPase activity of Herpetomonas sp. Intact cells were incubated for 1 h at room temperature with 500 μm DIDS or suramin in the presence or absence of 50 μm CrATP in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and 2 mm ATP. Data are means±s.e. of 3 determinations with different cell suspensions.

ADP also inhibited the ecto-ATPase of Herpetomonas sp. with apparent Ki of 1 mm and 40% maximal inhibition (not shown), comparable to the pattern described for Herpetomonas muscarum muscarum (Meyer-Fernandes et al. Reference Meyer-Fernandes, Saad-Nehme, Peres-Sampaio, Belmont-Firpo, Bisaggio, Do Couto, Fonseca De Souza, Lopes and Souto-Padron2004). The ADP and CrATP maximal inhibitions were not additive (Fig. 6).

Fig. 6. Effects of CrATP and ADP on the ecto-ATPase activity of Herpetomonas sp. Intact cells were incubated for 1 h at room temperature with 2 mm [γ-32P]ATP (400 cpm/nmol), in the presence of 200 μm CrATP, 2 mm ADP or both, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose and 2 mm MgCl2. Data are means±s.e. of 3 determinations with different cell suspensions.

The inhibition observed with CrATP on the ecto-ATPase of Herpetomonas sp. was also observed in Trypanosoma cruzi, Trypanosoma rangeli and Leishmania amazonensis (Fig. 7). In all cases the inhibitions were partial (especially obvious in the case of L. amazonensis) and with apparent Ki in the few micromolar range.

Fig. 7. Effect of increasing concentrations of CrATP on the ecto-ATPase activity of pathogenic trypanosomatids. Culture of intact cells of the trypanosomatids Leishmania amazonensis (▴), Trypanosoma cruzi (▪) and Trypanosoma rangeli (•) were washed with PBS and incubated in medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 3 mm MgCl2, 3 mm [γ-32P]ATP and increasing CrATP concentrations to estimate inhibition of ecto-ATPase activity. Activities in the absence of CrATP were (nmol Pi/108 cel×h cells) (▴) 62·3, (▪) 12·3, and (•) 16·3. Data are means±s.e. of 3 determinations with different cell suspensions.

DISCUSSION

CrATP has been used as a kinetic and structural tool because of its capacity to inhibit a series of enzymes that use the MgATP complex as substrate. The catalysis of most of the enzymes inhibited by CrATP involves transference of a phosphate group from ATP either to a second substrate (like glucose in hexokinase) or to the enzyme itself, forming a phosphoenzyme that is part of the catalytic cycle of the enzyme. P-ATPases are within this last case. With these enzymes CrATP sits at the ATP site and forms stable complexes with the proteins leading to an irreversible enzyme inhibition. Here we show that ecto-ATPases from different trypanosomatids are also sensitive to CrATP, though in a reversible manner. The reversibility suggests that ecto-ATPases do not form phosphorylated intermediates during catalysis. This is also supported by their insensitivity to vanadate (Attias et al. Reference Attias, Roitman, Camargo, Dollet and De Souza1988; Meyer-Fernandes, Reference Meyer-Fernandes2002; Alves-Ferreira et al. Reference Alves-Ferreira, Dutra, Lopes, Ferreira-Pereira, Scofano and Meyer-Fernandes2003).

The Herpetomonas sp. does not exhibit any Mg-dependent hydrolysis of ADP, indicating the absence of a true ecto-NTPDase2 activity. By chemical assays some Mg-independent ATP or ADP hydrolysis was detected, nevertheless they were insensitive to CrATP. The ecto-phosphatase activity was also almost unaffected by CrATP.

The apparent affinities for CrATP (Ki) of the ecto-ATPases from trypanosomatids are in the order of a few μm, lower than those usually obtained for the other known inhibitors like DIDS and suramin (Meyer-Fernandes et al. Reference Meyer-Fernandes, Dutra, Rodrigues, Saad- Nehme and Lopes1997; Berredo-Pinho et al. Reference Berredo-Pinho, Peres-Sampaio, Chrispim, Belmont-Firpo, Lemos, Martiny, Vannier-Santos and Meyer-Fernandes2001; Bernardes et al. 2001; de Jesus et al. 2002). The value of Ki decreased near 10-fold when a 10-fold lower substrate concentration was used, suggesting a true competitive mechanism. However, the inhibition of the ecto-ATPase activities in Herpetomonas, Leishmania and Trypanosoma cruzi were not total. The maximal inhibitions attained were 30–60% at 2 mm ATP and 40–50 μm CrATP, suggesting that CrATP is a universal inhibitor of trypanosomatid ecto-ATPase activities. Regarding Herpetomonas sp., inhibition was maintained constant at least up to 200 μm, which is 50 times the apparent Ki. Intriguingly, the values of maximal inhibition were different depending on substrate concentration. Incomplete inhibition was also observed with DIDS in Herpetomonas sp., T. cruzi (Bernardes et al. 2001) and with ADP in Herpetomonas muscarum muscarum (Alves-Ferreira et al. Reference Alves-Ferreira, Dutra, Lopes, Ferreira-Pereira, Scofano and Meyer-Fernandes2003).

The most plausible explanation for the pattern of partial inhibition is to consider the presence of 2 different enzymes, one sensitive to CrATP, which also has high ATP apparent affinity (Km=85 μm), and another less sensitive (or insensitive) to CrATP with low substrate apparent affinity (Km=3·5 mm). It is noteworthy that CrATP is considered to bind at ‘high affinity ATP binding-sites’ in enzymes as distinct as P-type ATPases (Vilsen and Andersen, Reference Vilsen and Andersen1992; Vilsen, Reference Vilsen1995; Einholm et al. Reference Einholm, Vilsen and Andersen2004; Moreira et al. Reference Moreira, Rios and Barrabin2005) and glycokinase (Danenberg and Cleland, Reference Danenberg and Cleland1975). These data, the absence of additive effects with ADP as inhibitor, and the reversible competitive inhibition observed here suggest that the CrATP binding site may be the catalytic site of (at least some) ecto-ATPases. A complex MgATP dependence for ecto-ATPase activity has also been described in Tritrichomonas foetus (Alves-Ferreira et al. Reference Alves-Ferreira, Dutra, Lopes, Ferreira-Pereira, Scofano and Meyer-Fernandes2003). In this parasite the activity increases with 2 kinetic components with respect to MgATP concentration, one with Km=30 μm and a second with Km=2 mm. Ecto-ATPases have been classified into several groups according to their phylogenetic relationships (Robson et al. Reference Robson, Sévigay and Zimmermann2006; Zimmermann, Reference Zimmermann1999). All of them share a number of highly conserved domains. Small differences in the primary structures are enough for a considerable impact on catalytic properties and to account for the differences observed between related ecto-ATPases enzymes (Asai et al. Reference Asai, Miura, Sibley, Okabayashi and Takeuchi1995; Nakaar et al. Reference Nakaar, Beckers, Polotsky and Joiner1998; Heine et al. Reference Heine, Braun, Heilbronn and Zimmermann1999). The alternative to 2 enzymes is the possibility of one enzyme in two different states of aggregation. It is known that ecto-NTPDase enzymes exist as homoligomers (Stout and Kirley, Reference Stout and Kirley1996; Lewis-Carl et al. Reference Lewis-Carl, Smith and Kirley1998; Wang et al. Reference Wang, Ou and Guidotti1998) and agents that disturb the degree of aggregation affect their enzymatic properties.

In protozoa, several hypothesis have been proposed for the function of ecto-ATPases. They include acquisition of adenosine from the media, necessary for normal growth, modulation of parasite infection and virulence, and involvement in cellular adhesion (Meyer-Fernandes, Reference Meyer-Fernandes2002). However, their function is still an open question and CrATP has potential as a valuable selective tool for a better understanding of properties and role of ecto-ATPases in the biology of parasites. Furthermore, the use of CrATP as a new inhibitor can help to select among distinct activities of ATP hydrolysis on the trypanonomatids surface. In this paper, CrATP unravelled the existence of an ecto-ATPase with a high apparent affinity for ATP on a trypanosomatid surface. Considering the low amount of extracellular ATP such an enzyme may be of physiological importance.

This work was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), PRONEX. O. C. M. was a recipient of a doctoral fellowship from CNPq and P. F. R. was a recipient of an undergraduate fellowship from CNPq. We thank Rosângela Rodrigues Ferreira for her excellent technical assistance.

References

REFERENCES

Alves-Ferreira, M., Dutra, P. M., Lopes, A. H., Ferreira-Pereira, A., Scofano, H. M. and Meyer-Fernandes, J. R. (2003). Magnesium-dependent ecto-ATP diphosphohydrolase activity in Herpetomonas muscarum muscarum. Current Microbiology 47, 265–271.CrossRefGoogle ScholarPubMed
Asai, T. and Suzuki, Y. (1990). Remarkable activities of nucleoside triphosphate hydrolase in the tachyzoites of both virulent and avirulent strains of Toxoplasma gondii. FEMS Microbiology Letters 60, 89–92.CrossRefGoogle ScholarPubMed
Asai, T., Miura, S., Sibley, L. D., Okabayashi, H. and Takeuchi, T. (1995). Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii. Journal of Biological Chemistry 270, 11391–11397.CrossRefGoogle ScholarPubMed
Attias, M., Roitman, I., Camargo, E. P., Dollet, M. and De Souza, W. (1988). Comparative analysis of the fine structure of four isolates of trypanosomatids of the genus Phytomonas. The Journal of Protozoology 35, 365–370.CrossRefGoogle Scholar
Bakker-Grunwald, T. and Parduhn, H. (1993). The Ca(2+)-ATPase activity of Entamoeba histolytica is exposed towards the medium and towards the lumen of intracellular vesicles. Molecular and Biochemichal Parasitology 57, 167–170.CrossRefGoogle ScholarPubMed
Barros, F. S., De Menezes, L. F., Pinheiro, A. A., Silva, E. F., Lopes, A. H., De Souza, W. and Meyer-Fernandes, J. R. (2000). Ectonucleotide diphosphohydrolase activities in Entamoeba histolytica. Archives of Biochemistry and Biophysics 375, 304–314.CrossRefGoogle ScholarPubMed
Bermudes, D., Peck, K. R., Afifi, M. A., Beckers, C. J. and Joiner, K. A. (1994). Tandemly repeated genes encode nucleoside triphosphate hydrolase isoforms secreted into the parasitophorous vacuole of Toxoplasma gondii. Journal of Biological Chemistry 269, 29252–29260.CrossRefGoogle ScholarPubMed
Bernardes, C. F., Meyer-Fernandes, J. R., Saad-Nehme, J., Vannier-Santos, M., Peres-Sampaio, C. E. and Vercesi, A. E. (2001). Effects of 4,4′-diisothyocyanatostilbene-2,2′-disulfonic acid on Trypanosoma cruzi proliferation and Ca(2+) homeostasis. The International Journal of Biochemistry & Cell Biology 32, 519–527.CrossRefGoogle Scholar
Berredo-Pinho, M., Peres-Sampaio, C. E., Chrispim, P. P., Belmont-Firpo, R., Lemos, A. P., Martiny, A., Vannier-Santos, M. A. and Meyer-Fernandes, J. R. (2001). A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence. Archives of Biochemistry and Biophysics 391, 16–24.CrossRefGoogle ScholarPubMed
Binderup, K., Watanabe, L., Polikarpov, I., Preiss, J. and Arni, R. K. (2000). Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber. Acta Crystallographica. Section D, Biological Crystallography 56, 192–194.CrossRefGoogle ScholarPubMed
Bisaggio, D. F., Peres-Sampaio, C. E., Meyer-Fernandes, J. R. and Souto-Padron, T. (2003). Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite-host cell interaction. Parasitology Research 91, 273–282.CrossRefGoogle ScholarPubMed
Camargo, E. (1999). Phytomonas and other trypanosomatid parasites of plants and fruit. Advances in Parasitology 42, 29–112.CrossRefGoogle ScholarPubMed
Camargo, E. P., Kastelein, P. and Roitman, I. (1990). Trypanosomatid parasites of plants (phytomonas). Parasitology Today 60, 22–25.CrossRefGoogle Scholar
Catarino, L. M., Serrano, M. G. Jr., Covazzana, M., Almeida, M. L., Kaneshima, E. K., Campaner, M., Jankevicius, J. V., Teixeira, M. M. G. and Itow-Jankevicius, S. (2001). Classification of trypanosomatids from fruits and seeds using morphological, biochemical and molecular markers revealed several genera among fruit isolates. FEMS Microbiological Letters 201, 65–72.CrossRefGoogle ScholarPubMed
Danenberg, K. D. and Cleland, W. W. (1975). Use of chromium-adenosine triphosphate and lyxose to elucidate the kinetic mechanism and coordination state of the nucleotide substrate for yeast hexokinase. Biochemistry 14, 28–39.CrossRefGoogle ScholarPubMed
de Jesus, J. B., de Sa Pinheiro, A. A., Lopes, A. H. and Meyer-Fernandes, J. R. (2002). An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence. Zeitschrift für Naturforschung [C] 57, 890–896.CrossRefGoogle ScholarPubMed
De Pierre, J. W. and Karnovsky, M. L. (1973). Plasma membranes of mammalian cells: a review of methods for their characterization and isolation. The Journal of Cell Biology 56, 275–303.CrossRefGoogle Scholar
De Pierre, J. W. and Karnovsky, M. L. (1974). Ecto-enzymes of the guinea pig polymorphonuclear leukocyte. I. Evidence for an ecto-adenosine monophosphatase, adenosine triphosphatase, and -p-nitrophenyl phosphates. Journal of Biological Chemistry 249, 7111–7120.Google ScholarPubMed
DePamphilis, M. L. and Cleland, W. W. (1973). Preparation and properties of chromium (3)-nucleotide complexes for use in the study of enzyme mechanisms. Biochemistry 12, 3714–3724.CrossRefGoogle Scholar
Desjeux, P. (2004). Leishmaniasis: current situation and new perspectives. Comparative Immunology, Microbiology and Infectious Diseases 27, 305–318.CrossRefGoogle ScholarPubMed
Dollet, M. (1984). Plant diseases caused by flagellate protozoa (Phytomonas). Annual Review of Phytopathology 22, 115–132.CrossRefGoogle Scholar
dos Passos Lemos, A., de Sa Pinheiro, A. A., de Berredo-Pinho, M., Fonseca de Souza, L., Motta, C. M., de Souza, W. and Meyer-Fernandes, R. (2002). Ectonucleotide diphosphohydrolase activity in Crithidia deanei. Parasitology Research 88, 905–911.CrossRefGoogle ScholarPubMed
Einholm, A. P., Vilsen, B. and Andersen, J. P. (2004). Importance of transmembrane segment M1 of the sarcoplasmic reticulum Ca2+-ATPase in Ca2+ occlusion and phosphoenzyme processing. Journal of Biological Chemistry 279, 15888–15896.CrossRefGoogle ScholarPubMed
Freymuller, E., Milder, R., Jankevicius, J. V., Jankevicius, S. I. and Camargo, E. P. (1990). Ultrastructural studies on the trypanosomatid Phytomonas serpens in the salivary glands of a phytophagous Hemipteran. The Journal of Protozoology 37, 225–229.CrossRefGoogle Scholar
Furuya, T., Zhong, L., Meyer-Fernandes, J. R., Lu, H. G., Moreno, S. N. and Docampo, R. (1998). Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages. Molecular and Biochemical Parasitology 92, 339–348.CrossRefGoogle ScholarPubMed
Handa, M. and Guidotti, G. (1996). Purification and cloning of a soluble ATP diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum). Biochemical and Biophysical Research Communications 218, 916–923.CrossRefGoogle ScholarPubMed
Heine, P., Braun, N., Heilbronn, A. and Zimmermann, H. (1999). Functional characterization of rat ecto-ATPase and ecto-ATP diphosphohydrolase after heterologous expression in CHO cells. European Journal of Biochemistry 262, 102–107.CrossRefGoogle ScholarPubMed
Jankevicius, J. V., Jankevicius, S., Campaner, M., Conchon, I., Maeda, L. A., Teixeira, M. M. G., Freymuller, S. and Camargo, E. P. (1989). Life cycle and culturing of Phytomonas serpens (Gibbs), a trypanosomatid parasite of tomatoes. The Journal of Protozoology 36, 265–271.CrossRefGoogle Scholar
Kirley, T. L. (1997). Complementary DNA cloning and sequencing of the chicken muscle ecto-ATPase. Homology with the lymphoid cell activation antigen CD39. Journal of Biological Chemistry 272, 1076–1081.CrossRefGoogle ScholarPubMed
Lafont, A. (1909). Sur la présence d'um parasite de la classe des flagellés dans le latex de l'Euphorbia pilulifera. Comptes Rendus des Séances de la Société de Biologie et de ses Filiales 66, 1011–1013.Google Scholar
Lewis-Carl, S. A., Smith, T. M. and Kirley, T. L. (1998). Cross-linking induces homodimer formation and inhibits enzymatic activity of chicken stomach ecto-apyrase. Biochemical and Molecular Biology International 44, 463–470.Google ScholarPubMed
Linnertz, H., Thönges, D. and Schoner, W. (1995). Na+/K(+)-ATPase with a blocked E1ATP site still allows backdoor phosphorylation of the E2ATP site. European Journal of Biochemistry 232, 420–424.CrossRefGoogle ScholarPubMed
Lowry, O. H. and Lopes, J. (1946). The determination of inorganic phosphate in the presence of labile phosphate esters. Journal of Biological Chemistry 162, 421–428.CrossRefGoogle ScholarPubMed
Meyer-Fernandes, J. R. (2002). Ecto-ATPases in protozoa parasites: looking for a function. Parasitology International 51, 299–303.CrossRefGoogle Scholar
Meyer-Fernandes, J. R., Dutra, P. M. L., Rodrigues, C. O., Saad- Nehme, J.and Lopes, A. H. C. S. (1997). Mg-dependent ecto-ATPase activity in Leishmania tropica. Archives of Biochemistry and Biophysics 341, 40–46.CrossRefGoogle ScholarPubMed
Meyer-Fernandes, J. R., Saad-Nehme, J., Peres-Sampaio, C. E., Belmont-Firpo, R., Bisaggio, D. F., Do Couto, L. C., Fonseca De Souza, A. L. F., Lopes, A. H. and Souto-Padron, T. (2004). A Mg-dependent ecto-ATPase is increased in the infective stages of Trypanosoma cruzi. Parasitology Research 93, 41–50.CrossRefGoogle ScholarPubMed
Moreira, O. C., Rios, P. F. and Barrabin, H. (2005). Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state. Biochimica et Biophysica Acta 1708, 411–419.CrossRefGoogle Scholar
Nakaar, V., Beckers, C. J., Polotsky, V. and Joiner, K. A. (1998). Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase. Molecular and Biochemichal Parasitology 97, 209–220.CrossRefGoogle ScholarPubMed
Pauls, H., Bredenbröcker, B. and Schoner, W. (1980). Inactivation of (Na++K+)-ATPase by chromium(III) complexes of nucleotide triphosphates. European Journal of Biochemistry 109, 523–533.CrossRefGoogle ScholarPubMed
Plesner, L. (1995). Ecto-ATPases: identities and functions. International Review of Cytology 158, 141–214.CrossRefGoogle ScholarPubMed
Raushel, F. M. and Cleland, W. W. (1977). Bovine liver fructokinase: purification and kinetic properties. Biochemistry 16, 2169–2175.CrossRefGoogle ScholarPubMed
Redman, C. A., Schineider, P., Mehlert, A. and Ferguson, A. J. (1995). The glycoinositol-phospholipids of Phytomonas. The Biochemical Journal 311, 495–503.CrossRefGoogle ScholarPubMed
Robson, S. C., Sévigay, J. and Zimmermann, H. (2006). The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance. Purinergic Signal 2, 409–430.CrossRefGoogle ScholarPubMed
Serpersu, E. H., Summitt, L. L. and Gregory, J. D. (1992). Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site. Journal of Inorganic Biochemistry 4, 203–215.CrossRefGoogle Scholar
Smith, T. M. Jr., Kirley, T. L. and Hennessey, T. M. (1997). A soluble ecto-ATPase from Tetrahymena thermophila: purification and similarity to the membrane-bound ecto-ATPase of smooth muscle. Archives of Biochemistry and Biophysics 337, 351–359.CrossRefGoogle Scholar
Soares de Medeiros, L. C. A., Moreira, B. L. M., Kildare, M., de Souza, W., Plattner, H., Hentschel, J. and Barrabin, H. (2005). A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp. Molecular and Biochemical Parasitology 140, 175–182.CrossRefGoogle Scholar
Stout, J. G. and Kirley, T. L. (1996). Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity. Biochemistry 35, 8289–8298.CrossRefGoogle ScholarPubMed
Vilsen, B. (1995). Structure-function relationships in the Ca(2+)-ATPase of sarcoplasmic reticulum studied by use of the substrate analogue CrATP and site-directed mutagenesis. Comparison with the Na+,K(+)-ATPase. Acta Physiologica Scandinavica. Supplementum. 624, 1–146.Google ScholarPubMed
Vilsen, B. and Andersen, J. P. (1992). CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum. Journal of Biological Chemistry 26, 25739–25743.CrossRefGoogle Scholar
Walseth, T. F. and Johnson, R. A. (1979). The enzymatic preparation of [alpha-32P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. Biochimica et Biophysica Acta 562, 11–31.CrossRefGoogle Scholar
Wang, T. F., Ou, I. and Guidotti, G. (1998). The transmembrane domains of ectoapyrase (CD39) affect its enzymatic activity and quaternary structure. Journal of Biological Chemistry 273, 24814–24821.CrossRefGoogle ScholarPubMed
World Health Organization (2002). Control of Chagas' Disease. WHO Technical Report Series No. 905, 1–109. World Health Organization, Geneva.Google Scholar
Zimmermann, H. (1999). Two novel families of ectonucleotidases: molecular structures, catalytic properties and a search for function. Trends in Pharmacological Science 20, 231–236.CrossRefGoogle Scholar
Figure 0

Fig. 1. Time-course (A) and effect of cell density (B) on the ecto-ATPase activity by intact cells of Herpetomonas sp. Cells were incubated for different periods of time (A) or for a fixed period of 1 h (B) at 37°C. The reaction medium contained 50 mm Tris–HCl buffer, pH 7·2, 2 mm Tris–ATP [γ-32P]ATP (sp. act 104 Bq/nmol ATP), 2 mm MgCl2 and 1 mg/ml of protein which corresponds to 2·2×108 cells/ml. Data are means±s.d. of 3 determinations with different cell suspensions.

Figure 1

Fig. 2. Effect of increasing concentrations of CrATP on the ecto-ATPase (A) and phosphatase (B) activities of Herpetomonas sp. (A) Intact cells were incubated for 1 h at room temperature, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm [γ-32P]ATP (4000 cpm/nmol), in the absence (○) or presence (•) of 2 mm MgCl2. In the case of absence of MgCl2, 300 μm EDTA was added. Data are means±s.e. of 3 determinations with different cell suspensions. The black square shows the reversibility of ecto-ATPase inhibition. Cells were incubated at room temperature with 200 μm CrATP as above and washed by centrifugation. Ecto-ATPase activity was assayed with 2 mm [γ-32P]ATP. Values representing 100% activities were 4·90±0·04 (○) and 79·5±15 (•) nmol Pi/108 cel×h, respectively. (B) Intact cells were incubated for 1 h, at room temperature, in medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and increasing concentrations of CrATP, in the presence of 5 mmp-nitrophenylphosphate (pNPP). Data are means±s.e. of 3 determinations with different cell suspensions. 100% activity was 94±10 nmol pNP/108 cel×h.

Figure 2

Fig. 3. ATP dependence of the CrATP inhibition of the ecto-ATPase activity. (A) Intact cells were incubated for 1 h at room temperature, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and increasing concentrations of CrATP, in the presence of (▪) 200 μm [γ-32P]ATP (4000 cpm/nmol) or (•) 2 mm [γ-32P]ATP (400 cpm/nmol). Data are means±s.e. of 3 determinations with different cell suspensions. Data were fitted by non-linear regression using the equation v=100−As/(1+Ki/[CrATP]), where v is enzyme activity, As represents the sensitive maximal activity (in %) and Ki the concentration of CrATP to inhibit half of the As. The activity at ‘infinite’ CrATP concentration, Ai=100−As, represents the percentage of insensitive activity. In (▪) 100% activity was 32·1±1·4 nmol Pi/108 cel×h cells and Ki=0·33±0·10 μm; in (•) 100% activity was 79·5±15 nmol Pi/108 cel×h and Ki=4·7±1·0 μm. (B) Activities measured in the absence (○) and in the presence (•) of 200 μm CrATP at different ATP concentrations. The difference between the curves (CrATP sensitive ecto-ATPase) was calculated (▴). The lines represent fitting of the data with the Michaelis-Menten equation (▴,○). The continuous line, representing the fit of the total activity, was calculated as the sum of the fitted sensitive and insensitive activities.

Figure 3

Fig. 4. Effect of DIDS (A) and suramin (B) on ecto-ATPase activity of Herpetomonas sp. Intact cells were incubated for 1 h at room temperature, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and increasing concentrations of (A) DIDS in the presence of (○) 200 μm or (•) 2 mm [γ-32P]ATP or (B) suramin in the presence of 2 mm [γ-32P]ATP. Data are means±s.e. of 3 determinations with different cell suspensions.

Figure 4

Fig. 5. Additive effects of DIDS or suramin and CrATP on ecto-ATPase activity of Herpetomonas sp. Intact cells were incubated for 1 h at room temperature with 500 μm DIDS or suramin in the presence or absence of 50 μm CrATP in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 2 mm MgCl2 and 2 mm ATP. Data are means±s.e. of 3 determinations with different cell suspensions.

Figure 5

Fig. 6. Effects of CrATP and ADP on the ecto-ATPase activity of Herpetomonas sp. Intact cells were incubated for 1 h at room temperature with 2 mm [γ-32P]ATP (400 cpm/nmol), in the presence of 200 μm CrATP, 2 mm ADP or both, in a medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose and 2 mm MgCl2. Data are means±s.e. of 3 determinations with different cell suspensions.

Figure 6

Fig. 7. Effect of increasing concentrations of CrATP on the ecto-ATPase activity of pathogenic trypanosomatids. Culture of intact cells of the trypanosomatids Leishmania amazonensis (▴), Trypanosoma cruzi (▪) and Trypanosoma rangeli (•) were washed with PBS and incubated in medium containing 50 mm Hepes-KOH buffer, pH 7·2, 65 mm KCl, 100 mm sucrose, 3 mm MgCl2, 3 mm [γ-32P]ATP and increasing CrATP concentrations to estimate inhibition of ecto-ATPase activity. Activities in the absence of CrATP were (nmol Pi/108 cel×h cells) (▴) 62·3, (▪) 12·3, and (•) 16·3. Data are means±s.e. of 3 determinations with different cell suspensions.