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Trypanosoma rangeli: Characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity
Abstract
In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.
... On the other hand, the presence of sodium in reaction media altered negatively the calculated Vmax for ATP hydrolysis ( Table 1). The effect of carbohydrates in the reaction media was tested since previous studies have described stimulation for Trypanosoma rangeli NTPDase (Fonseca et al., 2006). In fact, the presence of glucose or sucrose activated the rate of nucleotide hydrolysis as can be observed in Table 2. Substrate specificity was investigated and is presented in Table 3. ...
... Km remains unchanged, indicating that divalent cations are involved in catalysis, but not binding. This stimulatory effect was also observed for the T. brucei (de Souza Leite et al., 2007), T. rangeli (Fonseca et al., 2006) and T. cruzi (Meyer-Fernandes et al., 2004) NTPDases as well as other members of the NTPDAse family of enzymes (Knowles and Li, 2006) (Robson et al., 2006). ...
... Based on these evidences, researchers have previously investigated the role of carbohydrates on T. rangeli NTPDAse activity. Indeed, hydrolytic activity of T. rangeli NTPDase was increased by the presence of carbohydrates (Fonseca et al., 2006). In the present study we have also investigated the role of sugars into T. evansi NTPDase activity and have confirmed the observed activation. ...
Nucleoside triphosphate diphospho-hydrolases (NTPDases) catalyze the hydrolysis of several nucleosides tri and diphosphate playing major roles in eukaryotes including purinergic signaling, inflammation, hemostasis, purine salvage and host-pathogen interactions. These enzymes have been recently described in parasites where several evidences indicated their involvement in virulence and infection. Here, we have investigated the presence of NTPDase in the genome of Trypanosoma evansi. Based on the genomic sequence from Trypanosoma brucei, we have amplified an 1812 gene fragment corresponding to the T. evansi NTPDase gene. The protein was expressed in the soluble form and purified to homogeneity and enzymatic assays were performed confirming the enzyme identity. Kinetic parameters and substrate specificity were determined. The dependence of cations on enzymatic activity was investigated indicating the enzyme is stimulated by divalent cations and carbohydrates but inhibited by sodium. Bioinformatic analysis indicates the enzyme is a membrane bound protein facing the extracellular side of the cell with 98%identity to the T. brucei homologous NTPDase gene.
Copyright © 2015. Published by Elsevier Inc.
... Ecto-nucleoside triphosphate diphosphohydrolases are glycoproteins present in the plasma membrane with their active sites facing the external environment, which suggests that these enzymes may be involved in surface membrane interactions between parasites and their host cells. Ectonucleoside triphosphate diphosphohydrolases have been described in several protozoa parasites including Toxoplasma gondii [9][10][11][12][13][14][15], Tetrahymena thermophila [16], Leishmania sp, [17][18][19][20][21][22][23][24][25], Entamoeba histolytica [26], Acanthamoeba sp [27], Balamuthia mandrillaris [28], Trichomonas vaginalis [29][30][31], Trichomonas foetus [32], Trichomonas gallinae [33], Giardia lamblia [34], Crithidia deanei [35], Herpetomonas sp [36,37] and Trypanosoma sp [38][39][40][41][42][43][44]. These enzymes are divalent cation-dependent. ...
... These enzymes are divalent cation-dependent. In trypanosomatide parasites such as Leishmania tropica [21], Leishmania amazonensis [20], Crithidia deanei [35], Trypanosoma cruzi [40] and Trypanosoma rangeli [39], the ecto-ATPase activities are stimulated by magnesium and manganese, but not by calcium [20,21,40]. In Trypanosoma brucei the ecto-ATPase activity is stimulated by magnesium and manganese, and also by calcium and zinc [38]. ...
... Galactose was a good activator of T. cruzi ecto-ATPase [40] while fructose was a good activator of T. rangeli ecto-ATPase [39]. Similar findings were also observed with other protozoan parasites: galactose stimulated ecto-ATPase from Entamoeba histolytica [26], Tritrichomonas foetus [32], Trichomonas vaginalis [31], and Balamuthia mandrilaris [28] while mannose stimulated the ecto-ATPase from Acanthamoeba [27]. ...
Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases), also known as ecto-ATPases and/or ecto- apyrases, are integral membrane glycoproteins or soluble enzymes that are dependent on divalent cations. These ecto- enzymes are important ecto-nucleotidases that are characterized by the ability to hydrolyze nucleoside triphosphates and nucleoside diphosphates to the monophosphate form. The hydrolysis of nucleoside monophosphates to nucleosides such as adenosine may then be catalyzed by the action of ecto-5´nucleotidases. The present study reviews the sequential hy- drolysis of ATPADPAMPadenosine catalyzed by these ecto-enzymes from different trypanosomatids. These reactions participate in the salvage of purines in these parasites and simultaneously interfere with the establishment of in- fection and changes in the host immune response.
... T. rangeli also exhibits Mg 2ϩdependent ecto-NTPDase activity that is highest against ATP, although ADP and other NTPs may also be hydrolyzed. NTPDase activity is stimulated by a number of carbohydrates, which has led to the suggestion that NTPDase activity may have a role in adhesion to the intermediate insect host, as carbohydrates on insect salivary glands play a part in adhesion by Trypanosoma species (59). ...
... One explanation may be that in all three trypanosome species in which NTPDases have been studied, the enzymes hydrolyze ATP preferentially, resulting in liberation of ADP and transient platelet activation. Although preferential ATPase activity would provide ADP for activation of P2Y 1 and P2Y 12 receptors, ADP hydrolysis by these enzymes is nevertheless still relatively efficient (47,57,59). Again, more work is needed to clarify the contribution of NTPDases to platelet responses during infection. ...
... Unusually, Lpg1905 from L. pneumophila hydrolyzes only ATP/ADP and GTP/GDP and shows very limited activity against CTP/CDP and UTP/UDP (124). The NTPDases that do efficiently hydrolyze nonadenine nucleotides include those found in the apicomplexan parasites T. gondii (6) and N. caninum (4) and those found in T. vaginalis (40), T. foetus (80), the trypanosomes (47,57,59), and S. mansoni (53). In addition to their activity against NTPs, the NTPDases from T. gondii (6) other NDPs. ...
In humans, purinergic signaling plays an important role in the modulation of immune responses through specific receptors that recognize nucleoside tri- and diphosphates as signaling molecules. Ecto-nucleoside triphosphate diphosphohydrolases (ecto-NTPDases) have important roles in the regulation of purinergic signaling by controlling levels of extracellular nucleotides. This process is key to pathophysiological protective responses such as hemostasis and inflammation. Ecto-NTPDases are found in all higher eukaryotes, and recently it has become apparent that a number of important parasitic pathogens of humans express surface-located NTPDases that have been linked to virulence. For those parasites that are purine auxotrophs, these enzymes may play an important role in purine scavenging, although they may also influence the host response to infection. Although ecto-NTPDases are rare in bacteria, expression of a secreted NTPDase in Legionella pneumophila was recently described. This ecto-enzyme enhances intracellular growth of the bacterium and potentially affects virulence. This discovery represents an important advance in the understanding of the contribution of other microbial NTPDases to host-pathogen interactions. Here we review other progress made to date in the characterization of ecto-NTPDases from microbial pathogens, how they differ from mammalian enzymes, and their association with organism viability and virulence. In addition, we postulate how ecto-NTPDases may contribute to the host-pathogen interaction by reviewing the effect of selected microbial pathogens on purinergic signaling. Finally, we raise the possibility of targeting ecto-NTPDases in the development of novel anti-infective agents based on potential structural and clear enzymatic differences from the mammalian ecto-NTPDases.
... We also inferred that adenosine molecules could be transported into the parasite, probably via an adenosine transporter (De Koning et al., 1998). The physiological role of the ecto-ATPases in protozoa parasites is still unknown, but a possible involvement in parasite proliferation has been proposed (Meyer-Fernandes, 2002; Meyer-Fernandes et al., 2004; Fonseca et al., 2006).Fig. 7 shows that the Mg 2+ -dependent ATPase activity decreased during the time course of cell growth. ...
... The addition of CaCl 2 , ZnCl 2 , MgCl 2 , and MnCl 2 to the extracellular medium stimulated the ecto- ATPase activity (Fig. 1A ). The ecto-ATPase activity present in T. brucei brucei hydrolyses ATP, ITP, GTP, CTP, and UTP at high rates (Table 2) as also observed with the ecto-ATPase present on the surface of Trypanosoma cruzi (Meyer-Fernandes et al., 2004) and Trypanosoma rangeli (Fonseca et al., 2006 ). ADP was also recognized as substrate , indicating that this enzyme is an authentic nucleoside triphosphate diphosphohydrolase as described in other cells (Wang and Guidotti, 1996; Barros et al., 2000; Fonseca et al., 2006). ...
... The ecto-ATPase activity present in T. brucei brucei hydrolyses ATP, ITP, GTP, CTP, and UTP at high rates (Table 2) as also observed with the ecto-ATPase present on the surface of Trypanosoma cruzi (Meyer-Fernandes et al., 2004) and Trypanosoma rangeli (Fonseca et al., 2006 ). ADP was also recognized as substrate , indicating that this enzyme is an authentic nucleoside triphosphate diphosphohydrolase as described in other cells (Wang and Guidotti, 1996; Barros et al., 2000; Fonseca et al., 2006). Trypanosoma brucei brucei, as well as Leishmania amazonensis , are pathogens which cannot synthesize purines de novo (De Koning et al., 2000; Berredo-Pinho et al., 2001). ...
In this work we describe the ability of living cells of Trypanosoma brucei brucei to hydrolyze extracellular ATP. In these intact parasites there was a low level of ATP hydrolysis in the absence of any divalent metal (4.72+/-0.51 nmol Pi x 10(-7) cells x h(-1)). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 27.15+/-2.91 nmol Pi x 10(-7) cells x h(-1). This stimulatory activity was also observed when MgCl(2) was replaced by MnCl(2). CaCl(2) and ZnCl(2) were also able to stimulate the ATPase activity, although less than MgCl(2). The apparent K(m) for ATP was 0.61 mM. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid), as well as suramin, an antagonist of P(2) purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. Living cells sequentially hydrolyzed the ATP molecule generating ADP, AMP and adenosine, and supplementation of the culture medium with ATP was able to sustain the proliferation of T. brucei brucei as well as adenosine supplementation. Furthermore, the E-NTPDase activity of T. brucei brucei is modulated by the availability of purines in the medium. These results indicate that this surface enzyme may play a role in the salvage of purines from the extracellular medium in T. brucei brucei.
... Several ecto-enzymatic activities involved in the hydrolysis of phosphorylated substrates have been described in T. rangeli, including a Mg 2+ -dependent ecto-ATPase activity [57,58] and an ecto-phosphatase activity capable of hydrolyzing b-GP [50,59]. Table 1 shows the influence of several inhibitors on these activities. ...
... Other ecto-enzymes have been detected and characterized on the outer surface of the plasma membrane of this parasite, including a Mg 2+ -dependent ecto-ATPase [57,58] and an ectophosphatase that uses b-GP as a substrate [50,59]. To rule out the participation of these ecto-enzymes in the hydrolysis of PPi, we determined the sensitivity of each activity to classical phosphatase and pyrophosphatase inhibitors. ...
In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H+-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H+-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl2. In the presence of MgCl2, this activity was inhibited by millimolar concentrations of CaCl2. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.
... The optimum pH for ecto-ATPases described in other parasites such as Entamoeba histolytica ( Barros et al., 2000), Trichomonas vaginalis (Jesus et al., 2002a), Leishmania amazonensis (Berredo Pinho et al., 2001) and Trypanosoma rangeli ( Fonseca et al., 2006) lies on the alkaline range. Differently, in G. lamblia trophozoites, using a pH range from 5.6 to 8.4, where cells were alive throughout the time course of reaction, the Mg 2+-dependent ecto-ATPase activity was not modified (Fig. 1C), as also observed in Trypanosoma cruzi (Meyer-Fernandes et al., 2004) and Trypanosoma brucei brucei ( Leite et al., 2007) . ...
... Ecto-ATPases have been described in some protozoan parasites such as Acathamoeba sp. ( Sissons et al., 2004), Toxoplasma gondii ( Asai et al., 1995;Nakaar et al., 1998), E. histolytica ( Barros et al., 2000), T. thermophila ( Smith et al., 1997), Leishmania sp. (Berredo-Pinho et al., 2001;Meyer-Fernandes et al., 1997;Pinheiro et al., 2006), T. cruzi ( Fietto et al., 2004;Meyer-Fernandes et al., 2004), T. rangeli ( Fonseca et al., 2006), T. brucei brucei ( Leite et al., 2007), T. foetus ( Jesus et al., 2002) and T. vaginalis (de Jesus et al., 2002). The fact that virulent promastigotes of L. amazonensis has higher Mg 2+-dependent ecto-ATPase activity then avirulent promastigotes (Berredo-Pinho et al., 2001), as well as the invasive form of E. histolytica presents much higher Mg 2+dependent ecto-ATPase activity than noninvasive form ( Barros et al., 2000), support the speculative idea that the presence of the ecto-ATPase activity in trophozoites of G. lamblia could also be considered a pathogenesis marker for this cell. ...
In this work, we describe the ability of living trophozoites of Giardia lamblia to hydrolyze extracellular ATP. In the absence of any divalent cations, a low level of ATP hydrolysis was observed (0.78+/-0.08 nmol Pi x h(-1)x10(-6) cells). The ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. Half maximum stimulation of ATP hydrolysis was obtained with 0.53+/-0.07 mM. ATP was the best substrate for this enzyme. The apparent K(m) for ATP was 0.21+/-0.04 mM. In the pH range from 5.6 to 8.4, in which cells were viable, this activity was not modified. The Mg(2+)-stimulated ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A(1) (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The impermeant agent DIDS and suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases, decreased the enzymatic activity in a dose-dependent manner, confirming the external localization of this enzyme. Besides ATP, trophozoites were also able to hydrolyse ADP and 5 AMP, but the hydrolysis of these nucleotides was not stimulated by MgCl(2). Our results are indicative of the occurrence of a G. lamblia ecto-ATPase activity that may have a role in parasite physiology.
... While several non-selective chemical compounds have been used to inhibit E-NTPDases activity, strong and specific inhibitors are rare, most of them are nucleotide analogs (Zimmermann 2021a;Zimmermann, 2021b). The most wellknown CD39 inhibitors include an impermeant inhibitor, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) (Meyer-Fernandes et al., 1997;Bernardes et al., 2000), sodium azide (NaN3), suramin (Bisaggio et al., 2003;Fonseca et al., 2006), chelators (EDTA and EGTA) (Iqbal and Shah, 2018), ARL67156 (6-N, N-diethyl-D-b,g-dibromomethylene ATP) , 8-BuS-ATP derivatives (8-thiobutyladenosine 50triphosphate) (Gendron et al., 2000;Lecka et al., 2013) and BG0136 (1-naphthol-3, 6-disulfonic acid) (Gendron et al., 2002). Such inhibitors can increase ATP and ADP levels, causing the augmentation of purinergic signaling. ...
Belonging to the GDA1/CD39 protein superfamily, nucleoside triphosphate diphosphohydrolases (NTPDases) catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP) and inorganic phosphate (Pi). Several NTPDase isoforms have been described in different cells, from pathogenic organisms to animals and plants. Biochemical characterization of nucleotidases/NTPDases has revealed the existence of isoforms with different specificities regarding divalent cations (such as calcium and magnesium) and substrates. In mammals, NTPDases have been implicated in the regulation of thrombosis and inflammation. In parasites, such as Trichomonas vaginalis, Trypanosoma spp., Leishmania spp., Schistosoma spp. and Toxoplasma gondii, NTPDases were found on the surface of the cell, and important processes like growth, infectivity, and virulence seem to depend on their activity. For instance, experimental evidence has indicated that parasite NTPDases can regulate the levels of ATP and Adenosine (Ado) of the host cell, leading to the modulation of the host immune response. In this work, we provide a comprehensive review showing the involvement of the nucleotidases/NTPDases in parasites infectivity and virulence, and how inhibition of NTPDases contributes to parasite clearance and the development of new antiparasitic drugs.
... Radioligand binding identified two high-affinity plasmalemmal ATP receptors in Trypanosoma cruzi; the molecular nature of these receptors remains unknown [97]. Ecto-nucleotidases activity was detected in Trypanosoma [100], as well as in another member of the family, Leishmania [101]. ...
Purines and pyrimidines are indispensable molecules of life; they are fundamental for genetic code and bio-energetics. From the very early evolution of life purines have acquired the meaning of damage-associated extracellular signaller and purinergic receptors emerged in unicellular organisms. Ancestral purinoceptors are P2X-like ionotropic ligand-gated cationic channels showing 20-40% of homology with vertebrate P2X receptors; genes encoding ancestral P2X receptors have been detected in Protozoa, Algae, Fungi and Sponges; they are also present in some invertebrates, but are absent from the genome of insects, nematodes, and higher plants. Plants nevertheless evolved a sophisticated and widespread purinergic signalling system relying on the idiosyncratic purinoceptor P2K1/DORN1 linked to intracellular Ca 2+ signalling. The advance of metabotropic purinoceptors starts later in evolution with adenosine receptors preceding the emergence of P2Y nucleotide and P0 adenine receptors. In vertebrates and mammals the purinergic signalling system reaches the summit and operates throughout all tissues and systems without anatomical or functional segregation.
... The E-class of ecto-ATPases also includes diphosphohydrolases which, as the name indicates, hydrolyze both nucleotide tri-as well as di-phosphates. This enzyme, otherwise, exhibits the same chemical properties (alkaline pH range and requirement of divalent cations for activity) as ecto-ATPases (Fonseca et al. 2006) and is located in the plasma membrane of any pathogenic protozoan with its active site towards the extracellular environment (Pinheiro et al. 2006). However, golgi-located NTPDase has been shown to be virulent at insect stage whereas the secretory NTPDase can be dispensed, thus playing no role in virulence (Sansom et al. 2014). ...
The widespread role of ATP in humans has been characterized as a substrate for metabolism as well as other key processes occurring in the human system. Hence, the ATP pool may also be accessible to the invaders, including Leishmania donovani (a protozoan parasite), considering the fact that ATP is not scarce at the cellular location where the pathogen resides. The protozoan parasite survives in the human host by utilizing purines from its extracellular environment, due to its inability to synthesize purines de novo. The purines are accessible in the form of nucleoside triphosphates (NTPs), for example, adenosine (ADO) in the form of ATP molecules. These NTPs are processed by the ecto-nucleotidases in the transmembrane region of the parasite, which are then transported inside via nucleoside/nucleobase transporters belonging to the ENT family of transporters. Besides, the breakdown of NTPs by ecto-nucleotidases also yields inorganic phosphate (Pi) as by-product which is utilized by the parasite to maintain Pi homeostasis. These transporters have been characterized in protozoan parasites exhibiting homology in various species of Leishmania. Once inside the parasite, these purines (or their derivatives) are fluxed into purine metabolic pathways with the help of several cytosolic enzymes, prominently, adenosine deaminase, adenine amino hydrolase, phosphoribosyl transferases (APRT, HGPRT and XPRT) and adenosine kinase. This review outlines the predominant role of extracellular nucleotide metabolism and intracellular metabolic machinery in the containment of leishmanial infection. It also highlights the importance of inorganic phosphate transporter in relation to the purine transport. Graphical abstract: [Figure not available: see fulltext.].
... Cell membrane ecto-NTPDase are integral membrane glycoprotein, millimolar divalent cation-dependent, low specificity enzymes and hydrolyze all nucleoside triphosphates [24,27,30]. These enzymes were grouped into ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family [18]. These enzymes show many physiological functions such as (i) protection from lytic effects of extracellular ATP [41,45,50], (ii) regulation of ectokinase substrate concentration [14], (iii) termination of purinergic signaling [28,29], (iv) involvement in signal transduction [1,31,33], and (v) involvement in cellular adhesion [15,24,40]. ...
In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10(7) cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl2 stimulated the ATP hydrolysis to a greater extent compared with CaCl2 and ZnCl2. This activity was also observed when replaced by MnCl2. The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10(7) cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg(2+)-dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.
... The ecto-nucleotidases are located on the plasma membrane surface, where they hydrolyze extracellular nucleotides, and have been described in different cell types (Knowles, 2011;Meyer-Fernandes, 2002;Zimmermann, 2000Zimmermann, , 2012). Early reports have described the ecto-ATPase activities of different protozoan parasites, such as Toxoplasma gondii (Nakaar et al., 1998), Leishmania tropica (Meyer-Fernandes et al., 1997), L. amazonensis (de Almeida Marques-daSilva et al., 2008;Pinheiro et al., 2006), Leishmania braziliensis (Rezende-Soares et al., 2010), Entamoeba histolytica (Barros et al., 2000), Trypanosoma cruzi (Bernardes et al., 2000;Bisaggio et al., 2003;Fietto et al., 2004;Meyer-Fernandes et al., 2004), Trypanosoma rangeli (Fonseca et al., 2006) and Trypanosoma brucei (de Souza Leite et al., 2007). In this work, we demonstrated that L. amazonensis promastigotes resistant to 100 μM vinblastine showed increased levels of ecto-ATPase activity but not of other ectoenzyme activities. ...
Leishmania amazonensis is a protozoan parasite that induces mucocutaneous and diffuse cutaneous lesions upon infection. An important component in treatment failure is the emergence of drug-resistant parasites. It is necessary to clarify the mechanism of resistance that occurs in these parasites to develop effective drugs for leishmaniasis treatment. Promastigote forms of Leishmania amazonensis were selected by gradually increasing concentrations of vinblastine and were maintained under continuous drug pressure (resistant cells). Vinblastine-resistant L. amazonensis proliferated similarly to control parasites. However, resistant cells showed changes in the cell shape, irregular flagella and a decrease in rhodamine 123 accumulation, which are factors associated with the development of resistance, suggesting the MDR phenotype. The Mg-dependent-ecto-ATPase, an enzyme located on cell surface of Leishmania parasites, is involved in the acquisition of purine and participates in the adhesion and infectivity process. We compared control and resistant L. amazonensis ecto-enzymatic activities. The control and resistant Leishmania ecto-ATPase activities were 16.0 ± 1.5 nmol Pi × h(-1) × 10(-7) cells and 40.0 ± 4.4 nmol Pi × h(-1) × 10(-7)cells, respectively. Interestingly, the activity of other ecto-enzymes present on the L. amazonensis cell surface, the ecto-5' and 3'-nucleotidases and ecto-phosphatase, did not increase. The level of ecto-ATPase modulation is related to the degree of resistance of the cell. Cells resistant to 10 μM and 60 μM of vinblastine have ecto-ATPase activities of 22.7 ± 0.4 nmol Pi × h(-1) × 10(-7) cells and 33.8 ± 0.8 nmol Pi × h(-1) × 10(-7)cells, respectively. In vivo experiments showed that both lesion size and parasite burden in mice infected with resistant parasites are greater than those of L. amazonensis control cells. Furthermore, our data established a relationship between the increase in ecto-ATPase activity and greater infectivity and severity of the disease caused by vinblastine-resistant L. amazonensis promastigotes. Taken together, these data suggest that ecto-enzymes could be potential therapeutic targets in the struggle against the spread of leishmaniasis, a neglected world-wide public health problem.
... This could suggest that in this region, flagellates recognize and damage the basal membrane more easily, in order to cross it and reach the glandular epithelium cells. Fonseca et al. (2006) already demonstrated that lipophorins present in the hemolymph of R. prolixus influence on the improvement of the ecto-ATPase activity probably acting on the mechanism of tissues invasion of the parasites. ...
In vitro and in vivo experimental infection of Rhodnius robustus salivary glands by Trypanosoma rangeli: An ultrastructural approach of the initial process of invasion ABSTRACT The mechanism of invasion of triatomine salivary glands by Trypanosoma rangeli remains little known. To promote further information on the initial process of parasite invasion into glandular cells, an ultrastructural investigation was conducted after in vitro and in vivo infection of Rhodnius robustus salivary glands by a Brazilian strain of T. rangeli (SC-58). The Scanning Electron Microscopy images showed that the flagellates (trypomastigotes and/or epimastigotes) adhere themselves in the basal membrane of the glands mainly by the flagellum. It was also observed agglomerate of flagellates adhering in the basal membrane near the glandular duct, mostly epimastigotes. After one hour of infection, multiple clusters of flagellates spread out near to visible pores were recorded, an event not observed on the surface of the control gland. In Transmission Electron Microscopy it was observed the presence of various cross-sections and longitudinal figures of trypanosomes filling the gland lumen. The infected glands presented parasites similar to epimastigotes and trypomastigotes adhered to the microvilli and spreaded in the lumen. Under our experimental conditions flagellates (trypomastigotes and/or epimastigotes) appear to cause lesions in the basal membrane, crossing through it and invading the cells of glandular epithelium, in most cases using the tip of the flagellum.
... This could suggest that in this region, flagellates recognize and damage the basal membrane more easily, in order to cross it and reach the glandular epithelium cells. Fonseca et al. (2006) already demonstrated that lipophorins present in the hemolymph of R. prolixus influence on the improvement of the ecto-ATPase activity probably acting on the mechanism of tissues invasion of the parasites. ...
In vitro and in vivo experimental infection of Rhodnius robustus salivary glands by Trypanosoma rangeli: An ultrastructural approach of the initial process of invasion ABSTRACT The mechanism of invasion of triatomine salivary glands by Trypanosoma rangeli remains little known. To promote further information on the initial process of parasite invasion into glandular cells, an ultrastructural investigation was conducted after in vitro and in vivo infection of Rhodnius robustus salivary glands by a Brazilian strain of T. rangeli (SC-58). The Scanning Electron Microscopy images showed that the flagellates (trypomastigotes and/or epimastigotes) adhere themselves in the basal membrane of the glands mainly by the flagellum. It was also observed agglomerate of flagellates adhering in the basal membrane near the glandular duct, mostly epimastigotes. After one hour of infection, multiple clusters of flagellates spread out near to visible pores were recorded, an event not observed on the surface of the control gland. In Transmission Electron Microscopy it was observed the presence of various cross-sections and longitudinal figures of trypanosomes filling the gland lumen. The infected glands presented parasites similar to epimastigotes and trypomastigotes adhered to the microvilli and spreaded in the lumen. Under our experimental conditions flagellates (trypomastigotes and/or epimastigotes) appear to cause lesions in the basal membrane, crossing through it and invading the cells of glandular epithelium, in most cases using the tip of the flagellum.
... Other additions are indicated in the figure legends. For all experiments, the parasites were harvested from the culture medium by centrifugation at 1500g (4 °C) for 10 min and washed three times in a cold buffer solution containing 100 mM sucrose, 20 mM KCl and 50 mM Tris–HCl (pH 7.0) (Fonseca et al., 2006). ...
... Cell surface-located enzymes hydrolyzing extracellular nucleotides have been described in different cell types (Zimmermann, 2000; Zimmermann et al., 2012; Knowles, 2011). Early reports from our laboratory have described the ecto-ATPase activities of different protozoa, such as L. tropica (Meyer-Fernandes et al., 1997), L. amazonensis (Pinheiro et al., 2006), Entamoeba histolytica (Barros et al., 2000), T. cruzi (Meyer-Fernandes et al., 2004) and T. rangeli (Fonseca et al., 2006 ). Here, we show that heat shock promotes an increase in ecto-ATPase activity, but not in the activities of other ecto-enzymes that are present on the cell surface of T. cruzi . ...
... Neospora caninum, also an apicomplexan parasite, expresses a type I nucleoside triphosphate hydrolase [62]. Recently, ecto-ATPases have been identified in diverse trypanosomatids, including T. cruzi [63], T. rangeli [64], and T. brucei brucei [65]. While the physiological significance of some of these ecto-ATPases remains to be shown, virulent Leishmania amazonensis promastigotes can hydrolyze ATP by a Mgdependent ecto-ATPase more efficiently than avirulent promastigotes [66]. ...
The purinergic receptor, P2X7, has recently emerged as an important component of the innate immune response against microbial infections. Ligation of P2X7 by ATP can stimulate inflammasome activation and secretion of proinflammatory cytokines, but it can also lead directly to
killing of intracellular pathogens in infected macrophages and epithelial cells. Thus, while some intracellular pathogens
evade host defense responses by modulating with membrane trafficking or cell signaling in the infected cells, the host cells
have also developed mechanisms for inhibiting infection. This review will focus on the effects of P2X7 on control of infection by intracellular pathogens, microbial virulence factors that interfere with P2X7 activity, and recent evidence linking polymorphisms in human P2X7 with susceptibility to infection.
... Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), also named apyrases, ecto-ATPDases, E-type ATPDases or ecto-ATP-diphosphohydrolases, are integral membrane glycoproteins that are dependent on millimolar concentration of divalent cation and present a low specificity being able to hydrolyze all nucleoside di-and triphosphates (Kirley, 1997;Fonseca et al., 2006;Pinheiro et al., 2006Pinheiro et al., , 2008. More recently, these enzymes were grouped into the E-NTPDase family (Zimmermann, 2001). ...
Trypanosoma brucei brucei is the causative agent of animal African trypanosomiasis, also called nagana. Procyclic vector form resides in the midgut of the tsetse fly, which feeds exclusively on blood. Hemoglobin digestion occurs in the midgut resulting in an intense release of free heme. In the present study we show that the magnesium-dependent ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) activity of procyclic T. brucei brucei is inhibited by ferrous iron and heme. The inhibition of E-NTPDase activity by ferrous iron, but not by heme, was prevented by pre-incubation of cells with catalase. However, antioxidants that permeate cells, such as PEG-catalase and N-acetyl-cysteine prevented the inhibition of E-NTPDase by heme. Ferrous iron was able to induce an increase in lipid peroxidation, while heme did not. Therefore, both ferrous iron and heme can inhibit E-NTPDase activity of T. brucei brucei by means of formation of reactive oxygen species, but apparently acting through distinct mechanisms.
... It has been demonstrated that several pathogens present an enzymatic adenosine-generating apparatus to escape the host immune response (Tasca et al., 2003Tasca et al., , 2005 Borges et al., 2007; Marques-da-Silva et al., 2008; Pettengill et al., 2009; Thammavongsa et al., 2009; Kiffer-Moreira et al., 2010; Russo-Abrahão et al., 2011a,b). Trypanosomatids are incapable of synthesizing adenosine de novo; however, this nucleoside can be generated by ecto-nucleotidases via the sequential hydrolyzes of extracellular adenosine triphosphate (ATP) (Fonseca et al., 2006; Leite et al., 2007; Meyer-Fernandes et al., 2010 ). Our group has previously demonstrated that L. amazonensis possesses all of the enzymes that participate in sequential ATP hydrolysis (Berredo-Pinho et al., 2001; Pinheiro et al., 2006; Peres-Sampaio et al., 2008; de Souza et al., 2011). ...
Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.
... The presence of surface-located ATPases has been reported in many microorganisms (Zhong & Guidotti, 1999; Bernardes et al., 2000; De Jesus et al., 2002; Hopfe & Henrich, 2004; Sissons et al., 2004; Junior et al., 2005; Collopy-Junior et al., 2006). In several of these microbial models, including L. amazonensis (Berrêdo-Pinho et al., 2001), T. cruzi (Meyer-Fernandes et al., 2004; Santos et al., 2009) and Trypanosoma rangeli (Fonseca et al., 2006 ), maximum values of ATP hydrolysis were obtained at alkaline pHs, such as that currently described here. The specific functions of these enzymes are not fully known, but it has been demonstrated that they participate in many relevant biological processes (Zimmermann, 2001; Meyer-Fernandes, 2002). ...
In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.
... NTPDase and ecto-5′- nucleotidase activities have been identified in T. vaginalis, which may be involved in mechanisms related to host–parasite interactions controlling the levels of ATP and adenosine19202122. Further, NTPDase activities were characterized in other protozoan parasites: Achantamoeba [23]; Giardia lamblia [24]; Leishmania amazonensis [25]; Toxoplasma gondii [26]; Tritrichomonas foetus [27]; Trypanosoma cruzi282930; Trypanosoma brucei brucei [31] and Trypanosoma rangeli [32]. Considering that (i) Amaryllidaceae alkaloids present remarkable searching on cytotoxic activities and they are potential inhibitors of some enzymes1234567; (ii) purines and pyrimidines are released from dying cells, being early and universal indicators of cell damage33343536; and (iii) ATP acts as danger-associated molecular patterns (DAMPs) playing a role in cell signaling related to inflammation and immune responses [37], the aim of this study was to investigate the effect of lycorine and candimine on the T. vaginalis NTPDase and ecto-5′- nucleotidase activities. ...
Drug discovery from plants plays an important role in the pharmaceutical therapy field and the alkaloids lycorine and candimine are candidates for this purpose. Trichomonas vaginalis is a parasite that infects the human urogenital tract and causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ecto-nucleotidases including nucleoside triphosphate diphosphohydrolase (NTPDase) members, which hydrolyses extracellular ATP (adenosine triphosphate) and ADP (adenosine diphosphate), and ecto-5'-nucleotidase, which hydrolyses AMP (adenosine monophosphate), have been characterized in T. vaginalis. Because purine nucleotides are released from cells under physiological and stress conditions, the goal of this study was to evaluate the effect of lycorine and candimine on T. vaginalis NTPDase and ecto-5'-nculeotidase activities. The alkaloids (50 to 250microM) were tested against both long-term-grown and clinical isolates. Specific enzymatic activities were expressed as nmolPi released/min/mg protein. The effect of both alkaloids at NTPDase A and B expression levels was investigated. When the alkaloids were added directly to the reaction mixture, no effect on ATP, ADP or AMP hydrolysis was observed. NTPDase and ecto-5'-nucleotidase activities were strongly inhibited by candimine and lycorine on 24h-treated parasites. This effect was abolished when 24-treated parasites were innoculated in a culture medium without alkaloid. Transcript levels of NTPDase A or B were not altered by the alkaloids. Considering the cytotoxic and proinflammatory roles of ATP besides the anti-inflammatory effects of adenosine, the regulation of extracellular nucleotide levels could be relevant in increasing susceptibility of T. vaginalis to host immune response in the presence of lycorine and candimine.
... Another ecto-enzyme present on the external surface of T. rangeli is an ecto-ATPase [43]. Using ATP as a substrate, we tested the effects of H 2 O 2 on the ecto-ATPase activity in intact cells of T. rangeli. ...
As a protozoan parasite of hematophagous insects, Trypanosoma rangeli epimastigotes are exposed to reactive oxygen species during development in hosts. In this work, we investigated the role of H(2)O(2) as a modulator of the ecto-phosphatase activity present in living T. rangeli. We observed that H(2)O(2) inhibits ecto-phosphatase activities in the short and long epimastigote forms of T. rangeli. Ecto-phosphatase activity found in the short form was more sensitive than that found in the long form. Moreover, H(2)O(2) inhibited ecto-phosphatase activity of the short form in a dose-dependent manner and this inhibition was reversible after H(2)O(2) removal. This effect was not observed for T. rangeli ecto-ATPase, another ecto-enzyme present on the external surface of T. rangeli. Cysteine, beta-mercaptoethanol, and reduced glutathione were able to revert the enzyme inhibition promoted by H(2)O(2). Catalase and glutathione peroxidase stimulated this ecto-phosphatase activity, whereas superoxide dismutase was not able to modulate this activity. The ecto-phosphatase activity was also activated by FCCP and inhibited by oligomycin. It seems that H(2)O(2) plays a fundamental role in the regulation of cellular processes of these organisms. We showed, for the first time, that these parasites can produce H(2)O(2), and it is able to regulate ecto-phosphatase activity.
... Two types of receptors for ATP with different affinities were identified by radioligand binding assay in Trypanosoma cruzi; these receptors were suggested to play a role in the induction of parasitosis (Inverso et al. 1995). NTPDase activity has been characterized in Trypanosoma ( Fonseca et al. 2006, de Souza Leite et al. 2007. Leishmania releases nucleoside diphosphate kinase (NDPK), which prevents ATP-mediated cytolysis of macrophages ( Kolli et al. 2008). ...
Purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms. The evidence for purinergic signalling in plants, invertebrates and lower vertebrates is reviewed. Much is based on pharmacological studies, but important recent studies have utilized the techniques of molecular biology and receptors have been cloned and characterized in primitive invertebrates, including the social amoeba Dictyostelium and the platyhelminth Schistosoma, as well as the green algae Ostreococcus, which resemble P2X receptors identified in mammals. This suggests that contrary to earlier speculations, P2X ion channel receptors appeared early in evolution, while G protein-coupled P1 and P2Y receptors were introduced either at the same time or perhaps even later. The absence of gene coding for P2X receptors in some animal groups [e.g. in some insects, roundworms (Caenorhabditis elegans) and the plant Arabidopsis] in contrast to the potent pharmacological actions of nucleotides in the same species, suggests that novel receptors are still to be discovered.
... The presence of surface-located ATPases has been reported in many microorganisms (Zhong and Guidotti 1999;Bernardes et al. 2000;De Jesus et al. 2002;Sissons et al. 2004;Hopfe and Henrich 2004;Junior et al. 2005). In several of these microbial models, including Leishmania amazonensis (Berreˆdo-Pinho et al. 2001), Trypanosoma cruzi , and T. rangeli (Fonseca et al. 2006), maximum values of ATP hydrolysis were obtained at alkaline pHs, such as the currently described F. pedrosoi enzyme. The specific functions of these enzymes are not fully known, but it has been demonstrated that they participate in many relevant biological processes (Plesner 1995;Zimmermann 2001;Meyer-Fernandes 2002). ...
In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h−1 mg−1 mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h−1 mg−1) in the presence of 5 mM MgCl2, with values of V
max and apparent K
m for Mg-ATP2−corresponding to 541.9 ± 48.6 nmol Pi h−1 mg−1 cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg2+-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A1 (V-ATPases), and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg2+-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P2 purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.
... The identity and the function of ecto-ATPases have been reviewed and the nomenclature of ''E-type ATPases " was proposed to describe these enzymes (Plesner, 1995). Early reports from our laboratory have described ecto-ATPase activities in different protozoa, such as Leishmania tropica (Meyer-Fernandes et al., 1997), L. amazonensis (Pinheiro et al., 2006), Entamoeba histolytica (Barros et al., 2000), Trypanosoma cruzi (MeyerFernandes et al., 2004) and Trypanosoma rangeli (Fonseca et al., 2006). It has been reported that an increase in temperature induces, in vitro, differentiation from the promastigote to the amastigote form of Leishmania mexicana and the synthesis of heat-shock proteins (Alcina and Fresno, 1988). ...
In this work we demonstrated that promastigotes of Leishmania amazonensis exhibit an Mg-dependent ecto-ATPase activity, which is stimulated by heat shock. The Mg-dependent ATPase activity of cells grown at 22 and 28 degrees C was 41.0+/-5.2 nmol Pi/h x 10(7)cells and 184.2+/-21.0 nmol Pi/h x 10(7)cells, respectively. When both promastigotes were pre-incubated at 37 degrees C for 2h, the ATPase activity of cells grown at 22 degrees C was increased to 136.4+/-10.6 nmol Pi/h x 10(7) whereas that the ATPase activity of cells grown at 28 degrees C was not modified by the heat shock (189.8+/-10.3 nmol Pi/h x 10(7)cells). It was observed that Km of the enzyme from cells grown at 22 degrees C (Km=980.2+/-88.6 microM) was the same to the enzyme from cells grown at 28 degrees C (Km=901.4+/-91.9 microM). In addition, DIDS (4,4'-diisothiocyanatostilbene 2,2'-disulfonic acid) and suramin, two inhibitors of ecto-ATPases, also inhibited similarly the ATPase activities from promastigotes grown at 22 and 28 degrees C. We also observed that cells grown at 22 degrees C exhibit the same ecto-phosphatase and ecto 3'- and 5'-nucleotidase activities than cells grown at 28 degrees C. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat-shock effect on ecto-ATPase activity of cells grown at 22 degrees C were exposed at 37 degrees C for 2h. A comparison between the stimulation of the Mg-dependent ecto-ATPase activity of virulent and avirulent promastigotes by the heat shock showed that avirulent promastigotes had a higher stimulation than virulent promastigotes after heat stress.
... ATPase activity was found in association with various cell types in the circulating system , nervous and other tissues and shown to impact on several patho-pysiological processes [9] . Besides the members of the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDase)-family found in eukaryotes, other ecto-ATPases have been characterised in both eukaryotes and prokaryotes, such as in streptococci [10] and in several protozoan parasites including the genera Toxoplasma [11], Leishmania121314, Entamoeba [15], Trichomonas [16], Crithidia [17] and Trypanosoma181920. Common to all was the ecto-ATPase activity shown to be dependent on divalent cations and to be inhibited by the impermeant ATPase-inhibitor 4', 4', diisothiocyanostilbene 2'2'disul- fonic acid (DIDS) [15] and suramin (an antagonist of P 2 receptors and some ecto-ATPases) [21]. ...
In the facultative human pathogen Mycoplasma hominis, which belongs to the cell wall-less Mollicutes, the surface-localised substrate-binding domain OppA of the oligopeptide permease was characterised as the main ecto-ATPase.
With the idea that extra-cellular ATP could only be provided by the infected host cells we analysed the ATP release of HeLa cells after incubation with different preparations of Mycoplasma hominis: intact bacterial cells, the membrane fraction with or without OppA, recombinant OppA as well as an ATPase-deficient OppA mutant. Release of ATP into the supernatant of the HeLa cells was primarily determined in all samples lacking ecto-ATPase activity of OppA. In the presence of the ATPase inhibitor DIDS the amount of ATP in the OppA-containing samples increased. This increase was maximal after incubation with fractions containing OppA protein indicating that OppA is involved in ATP release and subsequent hydrolysis. Real-time PCR analyses revealed that the proliferation of HeLa cells is reduced after infection with M. hominis and flow cytometry experiments established that OppA induces greater apoptosis than necrosis of HeLa cells whereas the preservation of ecto-ATPase activity of OppA induces apoptosis.
The OppA induced ATP-release and -hydrolysis induced cell death of M. hominis infected HeLa cells was predominantly due to apoptosis rather than necrosis. Future work will elucidate whether the induction of apoptosis is indispensable for survival of these non-invasive pathogen.
... There is a possibility that the data shown in Figure 1 represent underestimates of the actual amount of ATP released from mouse astrocytes, because the activity of ecto-ATPases was not inhibited in the present experiments. To test this possibility, the effects of potent ecto-ATPase blockers, such as suramin and DIDS [55,56] as well as ARL 67156 [57], could be examined. However, suramin was found to almost completely inhibit the luciferin-luciferase reaction (HT Liu and Y Okada, unpublished), and DIDS was reported to block the maxi-anion channel activity [58]. ...
In the present study, we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress. We first examined the expression of mRNAs for proteins constituting possible ATP-releasing pathways that have been suggested over the past several years. In RT-PCR analysis using both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was seen for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not CFTR. Inhibitors of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 muM), an inhibitor of the maxi-anion channel, which has recently been shown to serve as a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.
... Okadaic acid and microcystin-LR, specific inhibitors of phosphoserine/threonine phosphatases family (Barford et al., 1998; Sommer et al., 2002), inhibited the Mg 2+ -dependent phosphatase activity in 23% and 83%, respectively. We also tested the Mg 2+ -dependent ecto-phosphatase activity of T. rangeli in the presence of some substrates to Mg 2+ -dependent ecto-nucleotidase activities recently described in T. rangeli (Fonseca et al., 2006). Adenine nucleotides (ATP, ADP, and 5 AMP) and adenosine were not able to inhibit the ecto-phosphatase activity (Table 1). ...
In this work, we characterized a Mg(2+)-dependent ecto-phosphatase activity present in live Trypanosoma rangeli epimastigotes. This enzyme showed capacity to hydrolyze the artificial substrate for phosphatases, p-nitrophenylphosphate (p-NPP). At saturating concentration of p-NPP, half-maximal p-NPP hydrolysis was obtained with 0.23mM Mg(2+). Ca(2+) had no effect on the basal phosphatase activity, could not substitute Mg(2+) as an activator and in contrast inhibited the p-NPP hydrolysis stimulated by Mg(2+). The dependence on p-NPP concentration showed a normal Michaelis-Menten kinetics for this phosphatase activity with values of V(max) of 8.94+/-0.36 nmol p-NP x h(-1) x 10(-7) cells and apparent K(m) of 1.04+/-0.16 mM p-NPP. Mg(2+)-dependent ecto-phosphatase activity was stimulated by the alkaline pH range. Experiments using inhibitors, such as, sodium fluoride, sodium orthovanadate and ammonium molybdate, inhibited the Mg(2+)-dependent ecto-phosphatase activity. Inorganic phosphate (Pi), a product of phosphatases, inhibited reversibly in 50% this activity. Okadaic acid and microcystin-LR, specific phosphoserine/threonine phosphatase inhibitors, inhibited significantly the Mg(2+)-dependent ecto-phosphatase activity. In addition, this phosphatase activity was able to recognize as substrates only o-phosphoserine and o-phosphothreonine, while o-phosphotyrosine was not a good substrate for this phosphatase. Epimastigote forms of T. rangeli exhibit a typical growth curve, achieving the stationary phase around fifth or sixth day and the Mg(2+)-dependent ecto-phosphatase activity decreased around 10-fold with the cell growth progression. Cells maintained at Pi-deprived medium (2 mM Pi) present Mg(2+)-dependent ecto-phosphatase activity approximately threefold higher than that maintained at Pi-supplemented medium (50 mM Pi).
Ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes located on the surface of the T. cruzi plasma membrane, which hydrolyze a wide range of tri-/-diphosphate nucleosides. In this work, we used previously developed genetically modified strains of Trypanosoma cruzi (T. cruzi), hemi-knockout (KO +/−) and overexpressing (OE) the TcNTPDase-1 gene to evaluate the parasite infectivity profile in a mouse model of acute infection (n = 6 mice per group). Our results showed significantly higher parasitemia and mortality, and lower weight in animals infected with parasites OE TcNTPDase-1, as compared to the infection with the wild type (WT) parasites. On the other hand, animals infected with (KO +/−) parasites showed no mortality during the 30-day trial and mouse weight was more similar to the non-infected (NI) animals. In addition, they had low parasitemia (45.7 times lower) when compared with parasites overexpressing TcNTPDase-1 from the hemi-knockout (OE KO +/−) group. The hearts of animals infected with the OE KO +/− and OE parasites showed significantly larger regions of cardiac inflammation than those infected with the WT parasites (p < 0.001). Only animals infected with KO +/− did not show individual electrocardiographic changes during the period of experimentation. Together, our results expand the knowledge on the role of NTPDases in T. cruzi infectivity, reenforcing the potential of this enzyme as a chemotherapy target to treat Chagas disease (CD).
ENTPDases are enzymes known for hydrolyzing extracellular nucleotides and playing an essential role in controlling the nucleotide signaling via nucleotide/purinergic receptors P2. Moreover, ENTPDases, together with Ecto-5´-nucleotidase activity, affect the adenosine signaling via P1 receptors. These signals control many biological processes, including the immune system. In this context, ATP is considered as a trigger to inflammatory signaling, while adenosine (Ado) induces anti-inflammatory response. The trypanosomatids Leishmania and Trypanosoma cruzi, pathogenic agents of Leishmaniasis and Chagas Disease, respectively, have their own ENTPDases named "TpENTPDases," which can affect the nucleotide signaling, adhesion and infection, in order to favor the parasite. Besides, TpENTPDases are essential for the parasite nutrition, since the Purine De Novo synthesis pathway is absent in them, which makes these pathogens dependent on the intake of purines and nucleopurines for the Salvage Pathway, in which TpENTPDases also take place. Here, we review information regarding TpNTPDases, including their known biological roles and their effect on the purinergic signaling. We also highlight the roles of these enzymes in parasite infection and their biotechnological applications, while pointing to future developments.
ATP-diphosphohydrolases (EC 3.6.1.5), also known as ATPDases, NTPases, NTPDases, E-ATPases or apyrases, are enzymes that hydrolyze a variety of nucleoside tri- and diphosphates to their respective nucleosides, being their activities dependent on the presence of divalent cations, such as calcium and magnesium. Recently, ATP-diphosphohydrolases were identified on the surface of several parasites, such as Trypanosoma sp, Leishmania sp and Schistosoma sp. In parasites, the activity of ATP-diphosphohydrolases has been associated with the purine recuperation and/or as a protective mechanism against the host organism under conditions that involve ATP or ADP, such as immune responses and platelet activation. These proteins have been suggested as possible targets for the development of new antiparasitic drugs. In this review, we will comprehensively address the main aspects of the location and function of ATP-diphosphohydrolase in parasites. Also, we performed a detailed research in scientific database of recent developments in new natural and synthetic inhibitors of the ATP-diphosphohydrolases in parasites.
Nucleoside Triphosphate Diphosphohydrolases (NTPDases) are enzymes that belong to the GDA1/CD39 protein superfamily. These enzymes catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP). Biochemical characterization of the nucleotidases/NTPDases from various types of cells, including those from plants, animals and pathogenic organisms, has revealed the existence of several isoforms with different specificities with respect to divalent cations (magnesium, calcium, manganese and zinc) and substrates. In mammals, the NTPDases play important roles in the regulation of thrombosis and inflammation. In parasites of the genus Leishmania, the causative agents of leishmaniasis, two NTPDase isoforms, termed NTPDase-1 and NTPDase-2 have been described. Independently of their cellular localization, whether cell-surface localized, secreted or targeted to other organelles, in some Leishmania species these NTPDases could be involved in parasite growth, infectivity and virulence. Experimental evidence has suggested that the hydrolysis of ATP and ADP by parasite ecto-nucleotidases can down-modulate the host immune response. In this context, the present work provides an overview of recent works that show strong evidence not only of the involvement of the nucleotidases/NTPDases in Leishmania spp infectivity and virulence but also of the molecular mechanisms that lead to the success of the parasitic infection.
The majority of living cells, tissues, and organisms have some form of sensitivity to ATP. ATP mediated signaling emerged at the dawn of evolution, most likely in a form of a primitive “danger” signal that flagged cell damage and the release of the intracellular content into the environment. Further evolution of purinergic signaling involved the appearance of specific systems of regulated ATP release from living cells, development of receptor systems and of ectonucleotidases that regulate ATP breakdown. This chapter provides a comprehensive account of the evolution of the purinergic signaling system.
Trypanosoma rangeli is a hemoflagellate protozoan parasite presenting an overlapping distribution with T. cruzi, the etiological agent of Chagas disease, in a wide geographical area in Latin America. Despite considered as non-pathogenic for man, T. rangeli shares several characteristics with T. cruzi such as vertebrate and invertebrate reservoirs, vectors and approximately half of the soluble antigenic determinants. Despite the importance of specific detection, little is know about T. rangeli in comparison to T. cruzi; several questions lack proper answers, including the controversies concerning T. rangeli´s taxonomic position. In this context, this short review attempted to congregate current aspects on the research of this parasite, approaching several subjects as life cycle, vector susceptibility, specific genes studies and genomic data.
Ecto-enzymes can be defined as membrane-bound proteins that have their active site facing the extracellular millieu. In trypanosomatids, the physiological roles of these enzymes remain to be completed elucidated; however, many important events have already been related to them, such as the survival of parasites during their complex life cycle and the successful establishment of host infection. This chapter focuses on two remarkable classes of ecto-enzymes: ecto-nucleotidases and ecto-phosphatases, summarizing their occurrence and possible physiological roles in Leishmania and Trypanosoma genera. Ecto-nucleotidases are characterized by their ability to hydrolyze extracellular nucleotides, playing an important role in purinergic signaling. By the action of these ecto-enzymes, parasites are capable of modulating the host immune system, which leads to a successful parasite infection. Furthermore, ecto-nucleotidases are also involved in the purine salvage pathway, acting in the generation of nucleosides that are able to cross plasma membrane via specialized transporters. Another important ecto-enzyme present in a vast number of pathogenic organisms is the ecto-phosphatase. These enzymes are able to hydrolyze extracellular phosphorylated substrates, releasing free inorganic phosphate that can be internalized by the cell, crossing the plasma membrane through a Pi-transporter. Ecto-phosphatases are also involved in the invasion and survival of parasite in the host cells. Several alternative functions have been suggested for these enzymes in parasites, such as participation in their proliferation, differentiation, nutrition and protection. In this context, the present chapter provides an overview of recent discoveries related to the occurrence of ecto-nucleotidase and ecto-phosphatase activities in Leishmania and Trypanosoma parasites.
In the first 20 years that followed the purinergic signalling hypothesis in 1972, most scientists were sceptical about its validity, largely because ATP was so well established as an intracellular molecule involved in cell biochemistry and it seemed unlikely that such a ubiquitous molecule would act as an extracellular signalling molecule. However, after the receptors for ATP and adenosine were cloned and characterized in the early 1990s and ATP was established as a synaptic transmitter in the brain and sympathetic ganglia, the tide turned. More recently it has become clear that ATP is involved in long-term (trophic) signalling in cell proliferation, differentiation and death, in development and regeneration, as well as in short-term signalling in neurotransmission and secretion. Also, important papers have been published showing the molecular structure of P2X receptors in primitive animals like Amoeba and Schistosoma, as well as green algae. This has led to the recognition of the widespread nature of the purinergic signalling system in most cell types and to a rapid expansion of the field, including studies of the pathophysiology as well as physiology and exploration of the therapeutic potential of purinergic agents. In two books, Geoffrey Burnstock and Alexej Verkhratsky have aimed at drawing together the massive and diverse body of literature on purinergic signalling. The topic of this first book is purinergic signalling in the peripheral and central nervous systems and in the individual senses. In a second book the authors focus on purinergic signalling in non-excitable cells, including those of the airways, kidney, pancreas, endocrine glands and blood vessels. Diseases related to these systems are also considered. © Springer-Verlag Berlin Heidelberg 2012. All rights are reserved.
In free-living Amoeba proteus (strain B), three forms of tartrate-sensitive phosphatase were revealed by using PAGE of supernatant of the ameba homogenate
obtained with 1% Triton X-100 or distilled water and subsequent staining of gels with 2-naphthyl phosphate as substrate (pH
4.0). The form with the highest mobility in the gel turned out to be sensitive to all tested phosphatase activity modulators.
Two other forms with the lower mobilities were completely or significantly inactivated not only by sodium L-(+)-tartrate,
but also by L-(+)-tartaric acid, sodium orthovanadate, ammonium vanadate, ammonium molybdate, EDTA, EGTA, O-phospho-L-tyrosine,
DL-dithiothreitol, H2O2, 2-mercaptoethanol, and ions of heavy metals—Fe2+, Fe3+, and Cu2+. Based on results of inhibitory analysis, lysosomal location in ameba cells, and wide substrate specificity of these two
forms, it was concluded that they belonged to non-specific acid phosphomonoesterases (AcP, EC 3.1.3.2). This AcP is suggested
to have both phosphomonoesterase and phosphotyrosylprotein phosphatase activities. Two ecto-phosphatases were revealed in
culture medium, in which amebae were cultivated. One of them was inhibited by the same reagent as the ameba tartrate-sensitive
AcP and seemed to be the AcP released into the culture medium in the process of exocytosis of the content of food vacuoles.
In the culture medium, apart from this AcP, another phosphatase was revealed; it was not affected by any tested inhibitors
of AcP and alkaline phosphatase. It cannot be ruled out that this phosphatase belongs to the ecto-ATPases found in many protists;
however, so far its ability to hydrolyze ATP has not yet been proven.
Nucleoside triphosphate diphosphohydrolases (NTPDases, GDA1_CD39 protein superfamily) play a diverse range of roles in a number of eukaryotic organisms. In humans NTPDases function in regulating the inflammatory and immune responses, control of vascular haemostasis and purine salvage. In yeast NTPDases are thought to function primarily in the Golgi, crucially involved in nucleotide sugar transport into the Golgi apparatus and subsequent protein glycosylation. Although rare in bacteria, in Legionella pneumophila secreted NTPDases function as virulence factors. In the last 2 decades it has become clear that a large number of parasites encode putative NTPDases, and the functions of a number of these have been investigated. In this review, the available evidence for NTPDases in parasites and the role of these NTPDases is summarized and discussed. Furthermore, the processes by which NTPDases could function in pathogenesis, purine salvage, thromboregulation, inflammation and glycoconjugate formation are considered, and the data supporting such putative roles reviewed. Potential future research directions to further clarify the role and importance of NTPDases in parasites are proposed. An attempt is also made to clarify the nomenclature used in the parasite field for the GDA1_CD39 protein superfamily, and a uniform system suggested.
In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3'AMP 10 times more than 5'AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3'nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3'nucleotidase activity was not observed on ecto-5'nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3'nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage-parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3'nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite-macrophage interaction.
Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-gamma and have smaller parasite load, after 8weeks of infection. Furthermore, after 1week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.
Lectin nucleotide phosphohydrolases (LNPs) are proteins which possess both apyrase catalytic activity (E.C. 3.6.1.5) and specific carbohydrate binding properties, and these are linked. To investigate the structural and functional properties for these proteins, two putative soluble plant LNPs, 4WC and 7WC (from white clover), and a putative soluble plant apyrase 6RG (from ryegrass) were chosen. Rabbit polyclonal antibodies for each plant apyrase were generated using highly purified, overexpressed recombinant 4WC or 7WC. In the case of 6RG, the C-terminal half of the protein constituted the best antigen for generating polyclonal antibodies. These antibodies showed high specificity and sensitivity. Active, recombinant 4WC and 6RG were overexpressed and purified using the baculoviral insect cell expression system (4WCbac-sup and 6RG:Hisbac), while 7WC (7WCcoli) was produced from E. coli inclusion bodies and subsequently refolded to give active enzyme. In course of overexpression, recombinant 4WC was localised in both the cellular fraction (4WCbac) and in the media supernatant (4WCbac-sup), while recombinant 6RG:Hisbac was only found in the cellular fraction (6RG:Hisbac) indicating that it was not secreted during insect cell growth. Secretion of 4WCbac was found to be dependent on N-glycosylation at N313 but not at N85 and elimination of one or both of these sites appeared to have little influence on apyrase activity. In addition, both 4WCbac and 6RG:Hisbac from the cellular fraction were fully functional. These results were compared with similar work performed on the animal ecto-apyrases which have different specific N-glycosylation sites required for secretion and activity. The 4WCbac-sup, 7WCcoli and 6RG:Hisbac proteins all showed apyrase activity, that is they catalysed the hydrolysis of nucleotide tri- and/or di-phosphates to their corresponding nucleotide monophosphates, and released inorganic phosphate in a divalent cation-dependent manner. However, the proteins exhibited different activities, substrate specificities, pH profiles and influence of inhibitors: 4WCbac-sup had a preference for NDPs with a pH optimum ≥9.5; 7WCcoli had a modest preference for NTPs with a pH optimum at 8.5; 6RG:Hisbac was almost exclusively an NTPase with a pH optimum at 6.5. Contrary to predictions based on phylogeny the proteins all bound to sulphated disaccharides and their catalytic activities were influenced both positively and negatively by the binding of specific chitosans. The data indicates that all three soluble plant apyrases investigated here were LNPs, in contrast to predictions from the literature. In order to pinpoint the regions responsible for determining substrate specificity and chitosan binding, chimeras were made using the N- and C-terminal halves of 4WC and 6RG. This resulted in fully functional reciprocal chimeras. Comparison of the apyrase activity for parents and chimeras, substrate specificity, optimal pH, influence of inhibitors on activity and effects of chitosans indicated that the C-terminus was responsible for determining substrate specificity. However, the influence of specific chitosans on the chimeras appeared to be dependent on both the N- and C-terminal portions of the proteins. In addition, chimeras were found to bind to the same sulphated disaccharides as the parent proteins. Preliminary crystal screening experiments were performed with highly purified preparations of 7WCcoli and 6RG:Hisbac. Under specific conditions 7WCcoli was found to form cube-like crystalline arrangements while 6RG:Hisbac formed hexagonal-like crystalline structures. A potential model for carbohydrate binding by LNPs is proposed and the possible biological roles of plant LNPs are discussed.
Legionella pneumophila is the predominant cause of Legionnaires disease, a severe and potentially fatal form of pneumonia. Recently, we identified an ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila, termed Lpg1905, which enhances intracellular replication of L. pneumophila in eukaryotic cells. Lpg1905 is the first prokaryotic member of the CD39/NTPDase1 family of enzymes, which are characterized by the presence of five apyrase conserved regions and the ability to hydrolyze nucleoside tri- and diphosphates. Here we examined the substrate specificity of Lpg1905 and showed that apart from ATP and ADP, the enzyme catalyzed the hydrolysis of GTP and GDP but had limited activity against CTP, CDP, UTP, and UDP. Based on amino acid residues conserved in the apyrase conserved regions of eukaryotic NTPDases, we generated five site-directed mutants, Lpg1905E159A, R122A, N168A, Q193A, and W384A. Although the mutations E159A, R122A, Q193A, and W384A abrogated activity completely, N168A resulted in decreased activity caused by reduced affinity for nucleotides. When introduced into the lpg1905 mutant strain of L. pneumophila, only N168A partially restored the ability of L. pneumophila to replicate in THP-1 macrophages. Following intratracheal inoculation of A/J mice, none of the Lpg1905 mutants was able to restore virulence to an lpg1905 mutant during lung infection, thereby demonstrating the importance of NTPDase activity to L. pneumophila infection. Overall, the kinetic studies undertaken here demonstrated important differences to mammalian NTPDases and different sensitivities to NTPDase inhibitors that may reflect underlying structural variations.
The presence and regulation by hyperosmotic medium of the ouabain-sensitive (Na++K+)ATPase of the Malpighian tubule cells of Rhodnius prolixus was investigated. The ouabain-sensitive (Na++K+)ATPase activity was 5.4 ± 0.5 nmol Pi x mg-1 x min-1. Vanadate 100 μM completely abolished this ATPase activity. In hyperosmotic medium, obtained by addition of 180 mM mannitol, the (Na++K+)ATPase activity was inhibited by 60%. When the cell lysates were preincubated in hyperosmotic medium for 30 minutes and the ATPase activity was assayed in isosmotic medium, the (Na++K+)ATPase activity was not modified. Addition of 50 ng/ml sphingosine, a protein kinase C inhibitor, abolished the inhibition of (Na++K+)ATPase activity in hyperosmotic medium. Furthermore, phorbol ester (TPA), an activator of protein kinase C, mimicked the effect of hyperosmotic shock on (Na++K+)ATPase activity. The increase in Ca concentration decreased the (Na++K+)ATPase activity by 60% in isosmotic medium, with maximal effect obtained in 10-6 M Ca. No effect was observed in hyperosmotic medium. The inhibitory effect of Ca2+ on the (Na++K+)ATPase was not reversed by sphingosine. These results indicate that the ouabain-sensitive (Na++K+)ATPase activity of the Malpighian tubule is regulated by both increasing Ca2+ concentration and by the osmolality of the medium by different and integrative ways.
Organisms and cellular systems are required to adapt to stress conditions like high temperature, often responding by accumulating organic solutes, such as sugars. This accumulation is associated with the effectiveness of these osmolytes in minimizing protein denaturation and membrane damage under stress conditions. In this work, we have studied the effect of sugars on the protection against thermal inactivation of mitochondrial F0F1-ATPase complex, in preparations of submitochondrial particles containing or depleted of inhibitor protein. We observed that after 15 min of pre-incubation at 70 °C of latent MgATP-submitochondrial particles (with inhibitor protein) in the presence of 1.5 м of sucrose or trehalose, or 3.0 ᴍ of glucose or fructose, about 80% of enzyme activity remained active. In the same conditions, but in the absence of sugars, the activity of the particles was completely abolished. Submitochondrial particles depleted of the inhibitor protein (AS-particles) were almost completely inactivated after 3 min of pre-incubation at 70 °C in the absence of sugars and more than 60% of the enzyme activity remained active when these particles were pre-incubated in the presence of sugars. In such condition, the enzyme acquires a more compact and heat-stable conformation. Sugars, as well as the inhibitor protein, inhibit reversibly F0F1-ATPase complex activity and protect this enzyme against inactivation by high temperature. Interestingly, the protection, promoted by sugars, of particles containing inhibitor protein is higher than of particles depleted of inhibitor protein, suggesting a synergism between sugar and inhibitor protein.
In the present work we characterized the secreted phosphatase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This housefly parasite hydrolyzed p-nitrophenylphosphate at a rate of 10.26 nmol Pi/mg protein/min. Classical inhibitors of acid phosphatases, such as sodium
orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate promoted a decrease in this phosphatase activity. When the parasites were
assayed in the presence of sodium tartrate, an inhibitor of Leishmania spp-secreted acid phosphatases, this activity was drastically diminished. Cytochemical analysis showed the localization of
this enzyme on the external surface and in the flagellar pocket of these parasites. Sodium tartrate inhibited this reaction,
confirming the biochemical data. Platelet-activating factor (PAF) inhibited the phosphatase activity determined in the supernatant
of living H. m. muscarum.
ABSTRACT In the present work ectophosphatase activities of three trypanosomatid parasites of plants were characterized using intact cells. Phytomonas françai, Phytomonas mcgheei, and Herpetomonas sp. hydrolyzed p-nitro-phenylphosphate at a rate of 5.40, 7.28, and 25.58 nmol Pi/mg of protein per min, respectively. Experiments using classical inhibitors of acid phosphatases such as sodium orthovanadate (NaVO(3)) and sodium fluoride (NaF) showed a decrease in phosphatase activities. Lithium fluoride (LiF) and aluminum chloride (AlCl(3)) were also used. Although AlCl3 had no effect, LiF was able to promote a decrease in the phosphatase activities. Interestingly, the inhibition caused by LiF was enhanced by the addition of AlCl3 during the reaction, probably due to the formation of fluoroaluminate complexes. This effect was confirmed by cytochemical analysis. In this assay, electron-dense cerium phosphate deposits were visualized on the external surface of the three parasites.
In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.
1. Using the incorporation of [methyl-3H]thymidine as a proliferation marker, the effects of various nucleosides and nucleotides on endothelial LLC-MK2 cells were studied. We found that ATP, ADP, AMP and adenosine in concentrations of 10 microM or higher stimulate the proliferation of these cells. 2. Inhibition of ecto-ATPase (EC 3.6.1.15), 5'-nucleotidase (EC 3.1.3.5) or alkaline phosphatase (EC 3.1.3.1) significantly diminished the stimulatory effect of ATP, indicating that the effect is primarily caused by adenosine and not by adenine nucleotides. Also, the effect depends only on extracellular nucleosides, since inhibition of nucleoside uptake by dipyridamole has no influence on proliferation. 3. Other purine nucleotides and nucleosides (ITP, GTP, inosine and guanosine) also stimulate cell proliferation, while pyrimidine nucleotides and nucleosides (CTP, UTP, cytidine and uridine) inhibit proliferation. Furthermore, the simultaneous presence of adenosine and any of the other purine nucleosides is not entirely additive in its effect on cell proliferation. At the same time any pyrimidine nucleoside, when added together with adenosine, has the same inhibitory effect as the pyrimidine nucleoside alone. 4. Apparently these proliferative effects are neither caused by any pharmacologically known P1-purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP, or D-myo-inositol 1,4,5-trisphosphate as second messenger, nor by extracellular Ca2+. 5. Therefore, we conclude that various purine and pyrimidine nucleosides can influence the proliferation of LLC-MK2 cells by acting on putative purinergic and pyrimidinergic receptors not previously described.
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote, Tetrahymena, at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme in Tetrahymena. It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+ or Mg2+ dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain, N-ethylmaleimide and p-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ecto-ATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein in Tetrahymena plasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase of Tetrahymena may play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.
Live Trypanosoma cruzi amastigotes hydrolyzed p-nitrophenylphosphate (PNPP), phospho-amino-acids and 32P-casein under physiologically appropriate conditions. PNPP was hydrolysed at a rate of 80 nmol.mg-1.h-1 in the presence of 5 mM MgCl2, pH 7.2 at 30 degrees C. In the absence of Mg2+ the activity was reduced 40% and we call this basal activity. At saturating concentration of PNPP, half-maximal PNPP hydrolysis was obtained with 0.22 mM MgCl2. Ca2+ had no effect on the basal activity, could not substitute Mg2+ as an activator and in contrast inhibited the PNPP hydrolysis stimulated by Mg2+ (I50 = 0.43 mM). In the absence of Mg2+ (basal activity) the stimulating half concentration (S0.5) for PNPP was 1.57 mM, while at saturating MgCl2 concentrations the corresponding S0.5 for PNPP for Mg(2+)-stimulated phosphatase activity (difference between total minus basal phosphatase activity) was 0.99 mM. The Mg-dependent PNPP hydrolysis was strongly inhibited by sodium fluoride (NaF), vanadate and Zn2+ but not by tartrate and levamizole. The Mg-independent basal phosphatase activity was insensitive to tartrate, levamizole as well NaF and less inhibited by vanadate and Zn2+. Intact amastigotes were also able to hydrolyse phosphoserine, phosphothreonine and phosphotyrosine but only the phosphotyrosine hydrolysis was stimulated by MgCl2 and inhibited by CaCl2 and phosphotyrosine was a competitive inhibitor of the PNPP hydrolysis stimulated by Mg2+. The cells were also able to hydrolyse 32P-casein phosphorylated on serine and threonine residues but only in the presence of MgCl2. These results indicate that in the amastigote form of T. cruzi there are at least two ectophosphatase activities, one of which is Mg2+ dependent and can dephosphorylate phospho-amino acids and phosphoproteins under physiological conditions.
For the first time, a soluble, dedicated E-type ecto-ATPase has been identified and purified. This fully soluble ecto-ATPase is released into the growth media of the single-celled eukaryote,Tetrahymena,at a constant rate over time (independent of the growth phase of the cells) and it has characteristics similar to those previously described for the membrane-bound ecto-enzyme inTetrahymena.It was purified by a combination of ion-exchange, size exclusion, and affinity chromatography and nondenaturing gel electrophoresis. Its molecular weight was determined to be approximately 66,000 Da by denaturing gel electrophoresis and approximately 69,000 Da by size exclusion chromatography of the native form. The purified soluble enzyme displays the general characteristics of a dedicated E-type ecto-ATPase such as Ca2+or Mg2+dependence, hydrolysis of ATP and other nucleoside triphosphates (but not nucleoside diphosphates) and insensitivity to common ATPase inhibitors (vanadate, azide, ouabain,N-ethylmaleimide andp-chloromercuriphenyl sulfonate). It was further shown to be immunologically similar (by polyclonal antibodies) to both the membrane-bound ectoATPase of chicken gizzard smooth muscle (66 kDa) and a 66-kDa protein inTetrahymenaplasma membranes. The ecto-ATPase enzyme activity was also shown to be present in both the body plasma membrane and ciliary plasma membrane fractions but the body membrane had slightly higher specific activities. We propose that this ecto-ATPase ofTetrahymenamay play a role in inactivating purinergic signals, such as in their chemorepulsion responses to external GTP and ATP. It may also play a minor role in extracellular nucleotide scavenging.
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker
of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca,Mg)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression
and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the
observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide
triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPJ to ADP and inorganic phosphate, is expressed upon cell activation, Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label, 5′-p-(fluorosulfonyl)benzoyl adenosine (5′-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide ant agonists results in the inhibition of antigen induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.
Apolipophorin III (apoLp-III) was isolated from the haemolymph of last instar larvae of Galleria mellonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-III from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-III was determined to be 18 077 Da using mass spectrometry. The heat treatment (90°C, 30 min) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-III without change in the molecular mass, indicating that a conformational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-III into last instar G. mellonella larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-III enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-III in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein. © 1997 Elsevier Science Ltd. All rights reserved
Trypanosoma rangeli uses the same range of insect vector species as the human pathogen T. cruzi, but unlike the latter species, T. rangeli is pathogenic to some of its reduviid bug vectors. In this paper some aspects of the hemocytic responses of Rhodnius prolixus, a susceptible host/vector of T. rangeli, are examined and compared with those of two nonsusceptible insect species, the American cockroach, Periplaneta americana, and the desert locust, Schistocerca gregaria. In contrast to Rhodnius, Periplaneta and Schistocerca clear trypanosomes injected into the hemocoel. By 4 hr postinoculation large numbers of nodules, multicellular aggregates, are formed in the hemocoels of the cockroaches and the locusts, and the number of nodules formed is dose dependent. Trypanosomes are observable in the nodules, mostly intracellularly within lysosomes, and are in various stages of degeneration. Nodules are formed by the reduviid bug's hemocytes but only after the infection has lasted a few days and when free trypanosomes are abundant.
The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a Km of 9.4±2.8 μM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine (Km=0.38±0.06 μM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5–15-fold following growth in purine-depleted medium. These increased rates were due to increased Vmax values (Km remained unchanged) and inhibited by cycloheximide (10 μM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine (Km∼0.2 μM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter (Km∼10 μM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.
The major insect lipoprotein, lipophorin, was previously shown to inhibit the in vitro adhesion of cockroach hemocytes. In the present study, we show that the large lipophorin subunit, apoLp-I, is the primary component that prevents adhesion of larval hemocytes of the greater wax moth, and that this inhibition is strongly Ca2+-dependent. We also provide immunological evidence for the existence of the small lipophorin subunit, apoLp-III, in the wax moth, and show that recombinant apoLp-III does not modulate cell adhesion. Finally, we report on the use of an artificial substrate, poly(2-hydroxyethyl methacrylate) as an adhesion inhibitor. Hemocytes do not adhere to the artificial substrate in either normal or Ca2+-free saline, and remain both viable and adherent after exposure. Our results therefore favour the use of this artificial substrate in physiological studies requiring nonadherent hemocyte populations in media containing Ca2+.
Lipophorin and lipophorin-deficient plasma, purified by KBr density ultracentrifugation, were tested for their ability to inhibit the adhesion of cockroach (Periplaneta americana) haemocytes in vitro. Haemocytes resuspended in purified lipophorin, either in the absence or presence of Ca2+, remained non-adherent and retained a discoid morphology in vitro for at least 30 min. In contrast, haemocytes incubated either with or without Ca2+ for 30 min in lipophorin-deficient plasma, bovine serum albumin, or saline alone adhered and flattened onto glass coverslips. This finding is important since to date it has been difficult to maintain insect haemocytes in a non-adhesive state in vitro.
Ecto-ATPase, a transmembrane enzyme that catalyzes the hydrolysis of extracellular ATP (ATPe) to ADP and inorganic phosphate, is expressed upon cell activation. Ecto-ATPase is inhibited by non-hydrolyzable ATP analogues, which are competitive inhibitors of the catalytic reaction, and the ATP analogue affinity label. 5'-p-(fluorosulfonyl)benzoyl adenosine (5'-FSBA), which irreversibly inhibits the catalytic activity. These nucleotide antagonists do not cross the cell membrane and are specific for ecto-ATPase in T cells, B cells and NK cells. Inhibition of ecto-ATPase by both reversible and irreversible nucleotide antagonists results in the inhibition of antigen-induced cytokine secretion and cytolytic activity of T cells. Likewise, granule release and cytolytic activity of NK cells as well as antibody secretion and spontaneous proliferation by B-cell hybridomas are inhibited. Inhibition of ecto-ATPase does not influence effector cell-target cell conjugate formation, but acts, in part, by regulating the influx of extracellular calcium that is necessary to maintain cellular activation. Thus, further elucidation of ecto-ATPase regulation and expression and its interaction with intracellular signal transduction events will provide a basis for understanding the role of the hydrolysis of ATPe by ecto-ATPase in lymphocyte effector function.
I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.
This article is intended to be the first in a series of detailed descriptions of the methods developed by the authors for experiments in skinned muscle cells and more specifically in skinned cardiac cells. This first section deals with the method used for calculating the composition of the perfusion solution containing multiple metals and ligands. Instead of presenting the Fortran IV computer program that they have routinely used, the authors found it preferable to develop programs adaptable to the newest generation of pocket calculators and to describe them in enough detail so that they can be used directly by investigators having no experience in programming. On the other hand, the mathematical rationale for these calculator programs is basically the same as that used in the Fortran computer program, and it is explained in such a way that it may also be useful to investigators using computers. Three programs are presented: 1) The first program computes the apparent stability constants of complexes between metals and ligands at specified pH values from absolute stability constants. This program also does the reverse operation: calculation of absolute stability constants from experimentally determined apparent stability constants. In addition, the program does all the preliminary calculations necessary for the computation of the ionic strength. The absolute stability constants for the most often used metal-ligand complexes are listed with a detailed justification for the selection of these data from among those available in the literature. The problems of the dependence of the stability constants upon temperature and ionic strength are also discussed. 2) The second program calculates the total concentrations of metals and ligands necessary to obtain specified free ion concentrations and a specified ionic strength. This corresponds to the usual problem that has to be solved when making up the experimental solutions. Accordingly, the method developed for mixing the chemicals employed on the most commonly used solutions is explained. Finally, a modification of this program which permits the total concentration of the major cation to be specified instead of the ionic strength is described. 3) The third program does the operation which is the reverse of that done in the second program: it calculates the free ion concentrations from specified total concentrations of metals and ligands. This permits one to convert the data obtained with one set of stability constants into those that would be obtained with another set of stability constants. The program is useful for a critical evaluation of the data and for a comparison of the results obtained by different authors. A correspondence between the free ion values obtained with the stability constants recommended in this article and those obtained with the stability constants used by other investigators is presented.
Endocytosis was studied at the ultrastructural level in different developmental forms of Trypanosoma cruzi after incubation of the parasites in the presence of gold-labeled proteins (albumin-Au, peroxidase-Au and transferrin-Au) and low-density lipoprotein (LDL-Au). Epimastigote (culture) forms actively ingested LDL and proteins. Initially, gold particles were seen adhering only to the cytostome and inside the flagellar pocket. In parasites incubated at 4 degrees C with transferrin-Au or peroxidase-Au, labeling was found only at these two sites, showing that receptor-mediated endocytosis occurs in both regions. In the cytoplasm, gold particles were seen only inside two different compartments: membrane-bound vesicles and reservosomes. Incubation of epimastigotes with acridine orange followed by fluorescence microscopy revealed intense orange staining, indicating that the reservosomes have an acidic pH. This staining was abolished after incubation of the parasites in the presence of ammonium chloride. These data confirm that this compartment is the site of accumulation of ingested lipids and proteins. Little intracellular labeling with transferrin-Au was found in in vitro-derived amastigotes and trypomastigotes (both lack reservosomes). However, although in amastigotes very few gold particles were seen bound to the cells, in trypomastigotes they were observed bound to the membrane that encloses the cell body, the flagellar pocket, and the flagellum, suggesting that the receptors are more abundant in this form.
Cell-mediated cytotoxicity involves a number of distinct mechanisms as well as the active participation of the target cell. Recently, several investigators have demonstrated that extracellular ATP can act as a cytotoxic effector.
1. The effects of some possible inhibitors of ectonucleotidases on the breakdown of extracellular ATP by strips of guinea-pig urinary bladder were investigated. 2. Suramin and ethacrynic acid (10 mM) both inhibited ATP breakdown significantly, and difluorodinitrobenzene (10 mM) inhibited it slightly whereas N-ethylmaleimide, adenosine 5'-(gamma-thiotriphosphate) (ATP-gamma-S) and reactive blue-2 (10 mM) were without effect. 3. The inhibitory effects of suramin on ATP breakdown were non-competitive. 4. Ethacrynic acid (1 mM) irreversibly inhibited contractions of the guinea-pig bladder induced by ATP, substance P, histamine, non-adrenergic, non-cholinergic nerve stimulation or KCl, whereas suramin (100 microM) had no inhibitory effect. 5. The results suggest that suramin might provide a starting point for the design of selective inhibitors of ectonucleotidases.
The formation of [32P]pyrophosphate from acetyl phosphate and [32P]orthophosphate was studied under conditions in which phosphate-metal salts or acetyl phosphate-metal salts precipitate. In the absence of precipitates in purely aqueous media, the initial rate constant of transphosphorylation (kobs) was extremely small and the formation of pyrophosphate was detected only in the presence of calcium. In various combinations, conditions such as high pH, high concentrations of reactants, and the presence of dimethyl sulfoxide caused three types of precipitates to form. In completely aqueous solution with an excess of orthophosphate, the crystals formed at high pH contained 3 mol of calcium for 2 mol of phosphate and they were poorly effective at promoting phosphorolysis. In the presence of dimethyl sulfoxide, the ratio of calcium to phosphate in the sediment was 1:1 and phosphorolysis proceeded at a high rate. In either solvent, an excess of acetyl phosphate caused precipitation of a complex containing 1 mol of acetyl phosphate to 1 mol of calcium. In aqueous media the rate constant of phosphorolysis increased with increasing precipitation of the acetyl phosphate-calcium complex. With destabilization of the anions by dimethyl sulfoxide the increase in kobs for a given amount of acetyl phosphate-calcium precipitated was 200-fold higher. Magnesium did not form precipitates and was ineffective in promoting transphosphorylation in completely aqueous media, either in the presence of excess phosphate or in the presence of excess acetyl phosphate. However, when precipitation of phosphate-magnesium or acetyl phosphate-magnesium was promoted by addition of dimethyl sulfoxide, phosphorolysis was observed with rate constants as high as those found in the presence of calcium. These results indicate that phosphorolysis of acetyl phosphate occurs at higher rates on the surface of solid structures, through highly specific interactions involving acetyl phosphate, orthophosphate, and divalent cations.
Various membrane ATPases have been tested for their sensitivity to bafilomycin A1, a macrolide antibiotic. F1F0 ATPases from bacteria and mitochondria are not affected by this antibiotic. In contrast, E1E2 ATPases--e.g., the K+-dependent (Kdp) ATPase from Escherichia coli, the Na+,K+-ATPase from ox brain, and the Ca2+-ATPase from sarcoplasmic reticulum--are moderately sensitive to this inhibitor. Finally, membrane ATPases from Neurospora vacuoles, chromaffin granules, and plant vacuoles are extremely sensitive. From this we conclude that bafilomycin A1 is a valuable tool for distinguishing among the three different types of ATPases and represents the first relatively specific potent inhibitor of vacuolar ATPases.
Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca2+ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca2+ exceeds 0.3 to 0.4 μm, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg2+ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca2+ concentration dependence of Ca uptake (K
0.5, 0.5 to 0.8 μm; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
A method is described for preparing /sup 32/P-labeled ATP of high
specific activity by an excbange reaction. The method is simple and requires
only substrates and enzymes that are available commercially. Hydrolysis of the
labeled ATP with heavy meromyosin indicates that 98 to 99% of the /sup 32/P is in
the 1 -phosphate group. (auth)
A surface membrane fraction isolated from Leishmania donovani promastigotes contained distinct 5'- and 3'-nucleotidase activities. These were distinguished from each other, and from a previously described surface membrane nonspecific acid phosphomonoesterase, on the basis of several properties. The 5'- and 3'-nucleotidases had p' optima of 6.5 and 8.5, respectively. In contrast to the 3'-nucleotidase, the 5'-nucleotidase was inhibited by both ammonium molybdate and fluoride ions; the latter inducing a biphasic response. Neither divalent cations nor chelators affected the 5'-enzyme activity whereas the 3'-enzyme was inactivated by EDTA. This inactivation was fully reversed following removal of the chelator, either by resuspension of the membranes in EDTA free medium or by addition of certain divalent cations in excess; Co2+ being the most effective. The 5'-nucleotidase had activity with both ribo- and deoxyribonucleotide substrates, whereas the 3'-nucleotidase did not hydrolyse deoxyribonucleotides.
Ultrastructural studies of the mechanism of penetration of the salivary gland of the reduviid bugRhodnius prolixus byTrypanosoma rangeli showed that trypanosomes from the haemocoele penetrate the outer “membranes” of the gland flagellum foremost, disrupting the inner layers, to pass between the muscle cells to reach the gland cell basement membrane. This latter is also penetrated flagellum foremost, the parasite invaginating the gland cell plasmalemma beneath, to create a vacuole in which the trypanosome crosses the gland cells to reach the central lumen, often only losing its containing vacuole just before leaving the cell.
The structure of the outer “membranes” surrounding the salivary gland appeared similar to, and often actually part of, the basement membrane of the gland cells. These outer “membranes” were found to enclose large numbers of multinuleate “giant form” trypanosomes, whose significance is as yet unknown, but could perhaps represent a stage in the life cycle of the parasite where genetic interchange could take place.
To study the role of parasite protein kinase C (PKC) activity in the uptake of Leishmania amazonensis by mononuclear phagocytes we treated the parasites with 12-O-tetradecanoyl phorbol-13-acetate (TPA) and/or sphingosine, before interaction assays. Promastigotes of Leishmania amazonensis were incubated with 20 ng/ml TPA and/or 50 ng/ml sphingosine before the interaction with murine peritoneal macrophages. The short treatment enhanced about 200% the parasite association with the host cells, whereas the sphingosine treatment reduced about 50% the promastigote binding, as did the prolonged TPA treatment. The binding of cells treated with both drugs was not significantly altered. Biochemical and cytochemical data indicate that the protein kinase C agonists TPA and sphingosine, respectively, increased and decreased acid phosphatase (AcP) activity. The addition of sodium tartrate, a secreted AcP inhibitor, suppressed the TPA enhancing effects, but did not affect the basal parasite binding observed in control cells. The supernatants of TPA-treated L. amazonensis promastigotes increased the parasite association by about the same extent as the TPA treatment, and this effect was also abolished by tartrate. Although TPA did not enhance the association of L. major, a species that does not secrete AcP, the supernatants of TPA-treated L. amazonensis increased it in a tartrate-sensitive manner. The results suggest that Leishmania amazonensis PKC activity may modulate its interaction with macrophages via secreted AcP.
Ecto-ATPases are ubiquitous in eukaryotic cells. They hydrolyze extracellular nucleoside tri- and/or diphosphates, and, when isolated, they exhibit E-type ATPase activity, (that is, the activity is dependent on Ca2+ or Mg2+, and it is insensitive to specific inhibitors of P-type, F-type, and V-type ATPases; in addition, several nucleotide tri- and/or diphosphates are hydrolysed, but nucleoside monophosphates and nonnucleoside phosphates are not substrates). Ecto-ATPases are glycoproteins; they do not form a phosphorylated intermediate during the catalytic cycle; they seem to have an extremely high turnover number; and they present specific experimental problems during solubilization and purification. The T-tubule Mg2+-ATPase belongs to this group of enzymes, which may serve at least two major roles: they terminate ATP/ADP-induced signal transduction and participate in adenosine recycling. Several other functions have been discussed and identity to certain cell adhesion molecules and the bile acid transport protein was suggested on the basis of cDNA clone isolation and immunological work.
Extracellular ATP, at micromolar concentrations, induces significant functional changes in a wide variety of cells and tissues. ATP can be released from the cytosol of damaged cells or from exocytotic vesicles and/or granules contained in many types of secretory cells. There are also efficient extracellular mechanisms for the rapid metabolism of released nucleotides by ecto-ATPases and 5'-nucleotidases. The diverse biological responses to ATP are mediated by a variety of cell surface receptors that are activated when ATP or other nucleotides are bound. The functionally identified nucleotide or P2-purinergic receptors include 1) ATP receptors that stimulate G protein-coupled effector enzymes and signaling cascades, including inositol phospholipid hydrolysis and the mobilization of intracellular Ca2+ stores; 2) ATP receptors that directly activate ligand-gated cation channels in the plasma membranes of many excitable cell types; 3) ATP receptors that, via the rapid induction of surface membrane channels and/or pores permeable to ions and endogenous metabolites, produce cytotoxic or activation responses in macrophages and other immune effector cells; and 4) ADP receptors that trigger rapid ion fluxes and aggregation responses in platelets. Current research in this area is directed toward the identification and structural characterization of these receptors by biochemical and molecular biological approaches.
In this study a possible association between (Ca(2+)-Mg2+)-ATPase activity and the neural cell adhesion molecule, NCAM, was investigated. The effects of various detergents on ATPase activity were evaluated, and it was found that solubilization of rat brain microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS, released a major fraction of the (Ca(2+)-Mg2+)-ATPase activity together with NCAM. Using different types of solid phase immunoadsorption it was shown that NCAM antibodies selectively isolated ATPase activity. Furthermore, agarose gel immunoelectrophoresis of solubilized brain microsomes followed by ATPase assay directly in the gel revealed ATPase activity associated with the NCAM immunoprecipitate. The NCAM-associated enzyme activity had a broad nucleoside triphosphate specificity and no strict selectivity for divalent cations, indicating that the enzyme probably is an ecto-ATPase. This raises a series of intriguing questions in relation to NCAM adhesive functions.
CD39, a 70- to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca2+,Mg2+)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.
The influence of enzymatic degradation on the neurotransmitter actions of ATP was studied using the ecto-ATPase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethyleneATP (ARL 67156). Field stimulation of the parasympathetic nerves innervating guinea-pig urinary bladder muscle strips (1-8 Hz for 20 s) produced characteristic biphasic contractions, the peak magnitudes of which were significantly increased by 29-32% by ARL 67156 (100 microM). A similar degree of enhancement was seen in the presence of atropine (1 microM), consistent with ARL 67156 acting to enhance the action of neuronally released ATP. The effects of ARL 67156 reversed rapidly on washout of the drug. Contractions evoked by exogenous ATP (100 microM) were also potentiated by ARL 67156 (100 microM), but those to the stable analogue alpha,beta-methyleneATP (5 microM) were unaffected. ARL 67156 (100 microM) also enhanced contractions to exogenous acetylcholine (1 microM) and histamine (3 microM), but this potentiation was abolished by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) (100 mciroM). It is concluded that when ATP acts as a neurotransmitter its postjunctional actions are attenuated by enzymatic degradation. ARL 67156 inhibits this breakdown.
In the present paper, we show the existence of a furosemide-sensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in cell lysates of Malpighian tubular cells from Rhodnius prolixus, which could be the biochemical expression of the Na(+)-pump. The main characteristics of this activity are: (1) K0.5 for Na+ = 1.49 +/- 0.18 mM, (2) Vmax = 2.8 +/- 0.1 nmol inorganic orthophosphate (Pi).mg prot-1.min-1, (3) it is fully abolished by 2 mM furosemide, (4)it is insensitive to ouabain concentrations up to 10(-2) M, (5) it is sensitive to the presence of vanadate in the incubation medium indicating it to be a P-type ATPase, and (6) it is stimulated by nanomolar concentrations of Ca2+ in the incubation medium.
It has become clear that members of both the E-NTPase and PDNPase protein family can act as ectoenzymes or exoenzymes, or both, or may even have an intracellular localization. The functional characterization of recently cloned members of the ecto-NTPase family is expected to widen the range of catalytic properties and cellular locations even further. The molecular diversity of ectonucleotidases now appears to be comparable with that of receptors for nucleotides. It seems likely that additional family members including splice variants remain to be discovered. The enormous molecular diversity and overlapping tissue distribution of the members of the E-NTPase and PDNP family make it difficult to assign specific functions to enzymes in individual tissues. However, first examples have been elaborated. The definition of their physiological substrates remains a major challenge. It is expected that the generation of heterozygous and homozygous knockout mice for each subtype followed by double-knockouts obtained by crossbreeding of single knockout mice will help to elucidate the functional roles of ectonucleotidases. These novel insights, together with the development of specific enzyme inhibitors or recombinant enzymes, or both, promise to offer important tools for basic research and therapy, and may provide new understanding of the control of cellular function at the local level.
Drosophila has appeared in recent years as a powerful model to study innate immunity. Several papers published in the past year shed light on the role of the three Rel proteins Dorsal, Dif and Relish in the regulation of antimicrobial peptide expression. In addition, the discovery that a blood serine protease inhibitor is involved in the control of the antifungal response indicates that Toll is activated upon triggering of a proteolytic cascade and does not function as a Drosophila pattern recognition receptor.
In this work, we describe the ability of living cells of Entamoeba histolytica to hydrolyze extracellular ATP. In these intact parasites, whose viability was determined by motility and by the eosin method, ATP hydrolysis was low in the absence of any divalent metal (78 nmol P(i)/h/10(5) cells). Interestingly, in the presence of 5 mM MgCl(2) an ecto-ATPase activity of 300 nmol P(i)/h/10(5) cells was observed. The addition of MgCl(2) to the extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 1.23 mM MgCl(2). Both activities were linear with cell density and with time for at least 1 h. The ecto-ATPase activity was also stimulated by MnCl(2) and CaCl(2) but not by SrCl(2), ZnCl(2), or FeCl(3). In fact, FeCl(3) inhibited both Mg(2+)-dependent and Mg(2+)-independent ecto-ATPase activities. The Mg(2+)-independent ATPase activity was unaffected by pH in the range between 6.4 and 8. 4, in which the cells were viable. However, the Mg(2+)-dependent ATPase activity was enhanced concomitantly with the increase in pH. In order to discard the possibility that the ATP hydrolysis observed was due to phosphatase or 5'-nucleotidase activities, several inhibitors for these enzymes were tested. Sodium orthovanadate, sodium fluoride, levamizole, and ammonium molybdate had no effect on the ATPase activities. In the absence of Mg(2+) (basal activity), the apparent K(m) for ATP(4-) was 0.053 +/- 0.008 mM, whereas at saturating MgCl(2) concentrations, the corresponding apparent K(m) for Mg-ATP(2-) for Mg(2+)-dependent ecto-ATPase activity (difference between total and basal ecto-ATPase activity) was 0.503 mM +/- 0.062. Both ecto-ATPase activities were highly specific for ATP and were also able to hydrolyze ADP less efficiently. To identify the observed hydrolytic activities as those of an ecto-ATPase, we used suramin, a competitive antagonist of P(2) purinoreceptors and an inhibitor of some ecto-ATPases, as well as the impermeant agent 4'-4'-diisothiocyanostylbenzene-2'-2'-disulfonic acid. These two reagents inhibited the Mg(2+)-independent and the Mg(2+)-dependent ATPase activities to different extents, and the inhibition by both agents was prevented by ATP. A comparison among the ecto-ATPase activities of three amoeba species showed that the noninvasive E. histolytica and the free-living E. moshkovskii were less efficient than the pathogenic E. histolytica in hydrolyzing ATP. As E. histolytica is known to have a galactose-specific lectin on its surface, which is related to the pathogenesis of amebiasis, galactose was tested for an effect on ecto-ATPase activities. It stimulated the Mg(2+)-dependent ecto-ATPase but not the Mg(2+)-independent ATPase activity.