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Characterization of an ecto-5′-nucleotidase (EC 3.1.3.5) activity in intact trophozoites of Trichomonas gallinae

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Characterization of an ecto-5′-nucleotidase (EC 3.1.3.5) activity in intact trophozoites of Trichomonas gallinae
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  Characterization of an ecto-5 0 -nucleotidase (EC 3.1.3.5) activityin intact trophozoites of   Trichomonas gallinae Fernanda Pires Borges a,b , Ba´rbara Gottardi b , Cristiane Stuepp b ,Anne Brandolt Larre´  b , Tiana Tasca c ,Geraldo Attilio De Carli b , Carla Denise Bonan d, * a  Departamento de Bioquı´ mica, Instituto de Cieˆ ncias Ba´ sicas da Sau´ de, Universidade Federal do Rio Grande do Sul, RS, Brazil b  Laborato´ rio de Parasitologia Clı´ nica, Departamento de Ana´ lises Clı´ nicas, Faculdade de Farma´ cia,Pontifı´ cia Universidade Cato´ lica do Rio Grande do Sul, Brazil c Centro Universita´ rio Metodista IPA, RS, Brazil d  Laborato´ rio de Neuroquı´ mica e Psicofarmacologia, Faculdade de Biocieˆ ncias,Pontifı´ cia Universidade Cato´ lica do Rio Grande do Sul, Avenida Ipiranga 6681, Pre´ dio 12, 90619-900 Porto Alegre, RS, Brazil Received 26 May 2006; received in revised form 19 July 2006; accepted 1 August 2006 Abstract This study describes the enzymatic properties of an ecto-5 0 -nucleotidase in  Trichomonas gallinae . The enzyme hydrolyzesnucleoside monophosphates at pH 7.2 and is activated by divalent cations, such as magnesium. Ecto-5 0 -nucleotidase activity wasinsensitive to levamisole, tetramisole (alkaline phosphatase inhibitors), and AMPCP (adenosine 5 0 -[ a , b -methylene]diphosphate), anecto-5 0 -nucleotidase inhibitor, whereas 0.1 mM ammonium molybdate (considered a potent inhibitor of 5 0 -nucleotidase activity)completelyinhibitedtheenzymeactivity.Theapparent K  M (Michaelisconstant)and V  max (maximumvelocity)valuesforMg 2+ -AMPwere 466  57 m M and 3.7  0.59 nmolPi/min/10 6 trichomonads, respectively. Considering that trichomonads lack the ability tosynthesize purines and pyrimidines  de novo , the presence of an ecto-5 0 -nucleotidase in intact trophozoites of   T. gallinae  could beimportantinregulatingtheextracellularnucleotidelevelsandgeneratingadenosine,essentialforthesurvivalstrategiesoftheparasite. # 2006 Elsevier B.V. All rights reserved. Keywords:  Adenosine; Ecto-5 0 -nucleotidase; Extracellular nucleotides;  Trichomonas gallinae 1. Introduction Purine nucleosides and nucleotides are mostlyreleased from cells which are stressed or anoxic, injuredand metabolically active (Chow et al., 1997). There isevidence that purines have cytotoxic properties (Stein-bergandDiVirgilio,1991).ExtracellularATPmayactasasignalingcompoundincytolyticmechanisms(Filippiniet al., 1990) and it is hydrolyzed to adenosine by a groupof ecto-enzymes named ecto-nucleotidases, whichincludes NTPDases (nucleoside triphosphate dipho-sphohydrolases) and ecto-5 0 -nucleotidase. NTPDase1(CD39, apyrase, ATP diphosphohydrolase) dephosphor-ylates ATP to AMP, which is hydrolyzed by the ecto-5 0 -nucleotidase (EC 3.1.3.5), resulting in adenosine(Zimmermann, 1996, 2001).Nucleoside monophosphate phosphohydrolase or 5 0 -nucleotidase, also known as CD73, is a glycosylatedprotein bound to the outer surface of the plasma www.elsevier.com/locate/vetparVeterinary Parasitology 143 (2007) 106–111* Corresponding author. Tel.: +55 51 3320 3500x4158;fax: +55 51 3320 3568. E-mail address:  cbonan@pucrs.br (C.D. Bonan).0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.vetpar.2006.08.006  membrane by a glycosylphosphatidylinositol anchor(Misumi et al., 1990). This enzyme acts on a variety of non-cyclic nucleoside monophosphates, such as AMP,CMP, UMP, IMP and GMP, inactivating them to therespective nucleosides and inorganic phosphate (Bian-chi and Spychala, 2003). Although the 5 0 -nucleotidasehas broad substrate specificity, AMP is considered to bethe major physiological substrate with  K  M  values in themicromolar range (Zimmermann, 1992, 1996). Theenzyme is variably expressed in a wide number of cellstypes under physiological and pathological conditions(Zimmermann, 1992).The presence of ecto-nucleotidases has beenreported on the surface of various parasites. Vasconce-los et al. (1993, 1996) demonstrated the presence of anATP diphosphohydrolase in  Schistosoma mansoni . AMg-dependent ecto-ATPase activity was described in  Leishmania tropica  (Meyer-Fernandes et al., 1997) and  Leishmania amazonensis  (Berreˆdo-Pinho et al., 2001).Furthermore, an ATP diphosphohydrolase and an ecto-5 0 -nucleotidase were described in  Trichomonas vagi-nalis  (Matos et al., 2001; Tasca et al., 2003). Ecto-5 0 -nucleotidase activity detected in intact trophozoites of two  T. vaginalis  isolates hydrolyzes nucleoside mono-phosphates and is activated by divalent cations, such asMg 2+ and Ca 2+ . The presence of an enzyme thathydrolyzes AMP to adenosine provides the nucleosiderequired for parasite growth, due to the lack of   de novo purine nucleotide synthesis among all trichomonadspecies (Heyworth et al., 1982, 1984; Wang, 1990;Munagala and Wang, 2003). Trichomonasgallinae isaflagellatedprotozoanwhichparasitizes a variety of birds all over the world. Thedomesticpigeon, Columbalivia ,istheprimaryhostofthisparasite. This trichomonad occurs in the upper digestivetract and in various organs of different avian groups(Stabler,1954;DeCarlietal.,1979).Thenormalsitesof  T.gallinae  are the mouth, pharynx, esophagus and crop,where they cause the formation of caseous lesions. Inpigeons, trichomoniasis is mainly a disease of youngbirds, causing serious losses among these birds. Theprotozoan is the causative agent of canker in pigeons,causingavarietyofpathologicmanifestationsdependingontheparasiteisolateandtheinfectedbirdspecies(Baker,1986;CooperandPetty,1988).Thevirulentisolatesmaycause lesions in the upper digestive tract of birds, whichallow the pathogen to enter the circulatory system, gainaccess to the liver, lungs, heart and pancreas, leading tohost death. Indeed, avian trichomoniasis has been consi-deredasignificanteconomiclossinchickensandturkeys.Taking into account that  T. gallinae  is a seriousveterinary disease pathogen, it is important to inves-tigate the biochemical aspects of this parasite thatcontribute to understanding features related to host–parasite interaction. The present study describes theproperties of an ecto-5 0 -nucleotidase activity in intacttrophozoites of   T. gallinae . 2. Materials and methods 2.1. Parasite culture The T.gallinae isolate,TG7,fromtheupperdigestivetractofdomesticpigeons, C.livia ,wasusedinthisstudy.Trichomonads were axenically cultured  in vitro  intrypticase-yeast extract-maltose (TYM) medium (Dia-mond, 1957) without agar (pH 7.2) supplemented with10% (v/v) inactivated bovine serum, without antibiotics(Stableretal.,1964;TascaandDeCarli,1999),at37  8 C.Viability of the isolates was maintained by storing theminliquidnitrogen(  196  8 C)with5%dimethylsulfoxide(DMSO) (Honigberg et al., 1965). Trichomonads fromthe logarithmic phase of growth were collected bycentrifugation at 750  g  for 5 min. The parasites werethen washed three times with 0.9% (w/v) NaCl solution,countedwithahaemocytometerandadjustedtoadensityof 4  10 6 organisms/mL. All samples were run intriplicate, with results achieved in at least three differentparasites suspensions. All organisms were viable basedon motility, assessed before and after incubations. Theviability was not affected by incubation conditions. 2.2. Enzyme assays After preparing the parasite samples, the optimumconditions for nucleotide hydrolysis were determined.Intact trophozoites of   T. gallinae  (10 6 trichomonads/ mL) were added to the reaction mixture containing50 mM Tris buffer (pH 7.2) and 3.0 mM MgCl 2 . Thesamples were preincubated for 5 min at 37  8 C in thereaction mixture. The reaction was initiated by theaddition of substrate AMP to a final concentration of 3.0 mM. After 15 min, the reaction was stopped byadding 200  m L 10% trichloroacetic acid. The sampleswere chilled on ice before assaying for the release of inorganic phosphate (Chan et al., 1986). Incubationtimes and parasite density were chosen in order toensure the linearity of the reactions. Controls with theaddition of the intact cells after mixing trichloroaceticacid were used to correct non-enzymatic hydrolysis of substrates and the averages of control values weresubtracted from the test samples. All enzyme assayswere run in triplicate. Specific activity is expressed asnmol of Pi/min/10 6 trichomonads. F.P. Borges et al./Veterinary Parasitology 143 (2007) 106–111  107  2.3. Statistical analysis Statistical analysis was conducted by Student’s  t   testor one-way analysis of variance (ANOVA), consideringa level of significance of 5%. 3. Results 5 0 -Nucleotidase activity has been described inbacteria, plant cells and in various vertebrate tissues(Zimmermann, 1992). In this study, an enzyme withcharacteristics of an ecto-5 0 -nucleotidase was detectedin intact trophozoites of   T. gallinae.  The time course forAMP hydrolysis was linear up to 15 min in the presenceof Mg 2+ . The product formation was linear in the rangeof 0.8–2.0  10 6 trichomonads/mL (data not shown).AMP hydrolysis was activated in the presence of divalent cations (Fig. 1) at the concentrations of 3.0, 5.0and 8.0 mM. In the presence of Mg 2+ plus 5.0 mMEDTA, there was a significant decrease of AMP hydro-lysis. The concentration of 3.0 mM magnesium, whichisaclassicalactivatorof5 0 -nucleotidase,waschosenforsubsequent enzyme assays.Ecto-5 0 -nucleotidase is an enzyme with a broadsubstrate specificity for nucleoside monophosphates,with preference for AMP (Zimmermann, 1996). Intacttrophozoites of   T. gallinae  hydrolyzed all nucleosidemonophosphates tested (CMP, GMP, UMP) at a lowerrate than AMP (Table 1). To avoid the influence of pyrophosphatase,pyrophosphate,whichisasubstrateforthis enzyme, was incubated with the intact organisms.The presence of pyrophosphatase can be excludedbecause there is no significant hydrolysis of pyropho-sphate in these assay conditions (Table 1).AMP hydrolysis was determined at substrate con-centrations in the range of 200–3000 m M. Enzymeactivity increased with increasing concentrations of thenucleotide (Mg 2+ fixedat3.0 mM with different concen-trations of AMP) (Fig. 2).  K  M  (Michaelis constant) and V  max (maximumvelocity)valuesinintacttrophozoitesof  T. gallinae  were estimated from the Lineweaver–Burk plots with three different enzyme preparations (Fig. 2).The apparent  K  M  and  V  max  values for Mg 2+ -AMP were466  57 m M (mean  S.D.) and 3.7  0.59 nmolPi/ min/10 6 trichomonads (mean  S.D.), respectively.To discard the influence of alkaline phosphatase onAMP hydrolysis the classical inhibitors of this enzyme,levamisole and tetramisole, were tested. Both com-pounds,testedtoafinalconcentrationof1.0 mM,hadnoeffect upon AMP hydrolysis in intact trichomonads(Table2).AMPCP(adenosine5 0 -[ a , b -methylene]dipho-sphate) and ammoniun molybdate, which are known 5 0 -nucleotidase inhibitors, were also tested. The enzymeactivity was dramatically inhibited in the presence of  F.P. Borges et al./Veterinary Parasitology 143 (2007) 106–111 108Fig. 1. Effect of MgCl 2  and CaCl 2  concentration on ecto-5 0 -nucleo-tidase activity in intact trophozoites of   Trichomonas gallinae . Closedbars and open bars represent 5 0 -nucleotidase activity in presence of MgCl 2  and CaCl 2 , respectively. Incubation conditions were describedin Section 2. The control group was incubated without the addition of cation. Bars represent the means  S.D. for three experiments, usingdifferent trophozoites suspensions.  ** Significant difference fromactivity in the presence of MgCl 2  3.0 mM (  p < 0.01).  * Significantdifference from activity in the presence of CaCl 2  3.0 mM (  p < 0.05).Table 1Substrate specificity of ecto-5 0 -nucleotidase from intact cells of   T.gallinae Substrate Relative activityAMP 1.00  0.02CMP 0.87  0.14GMP 0.68  0.10UMP 0.60  0.05PPi 0.058  0.01Data represents means  S.D. of at least three experiments. ControlAMP hydrolysis was 1.99  0.04 nmolPi/min/10 6 trophozoites.Results were expressed as percentage of control activity. The sub-strates were used at 3.0 mM, with MgCl 2  3.0 mM.Fig. 2. Effect of different concentrations of substrate (200–3000 m M)on AMP hydrolysis in intact trophozoites of   T. gallinae . All experi-ments used fixed 3.0 mM Mg 2+ with variable concentrations of nucleotide. Data represents mean  S.D. of three different experi-ments, each in triplicate.  1.0 mM ammonium molybdate. However, AMPCP didnot promote significant changes on AMP hydrolysis.These results suggest that AMP hydrolysis is due to anecto-5 0 -nucleotidase in intact trophozoites of   T. gallinae . 4. Discussion Theproteinson the cellsurface oftrichomonads playa major role in cytoadhesion, host–parasite interaction,nutrients acquisition and in the protection from thecytolytic effects (Petrin et al., 1998). The results of thepresent study demonstrate an ecto-5 0 -nucleotidase inintact trophozoites of   T. gallinae . This enzyme was notdependent of divalent cations, but the activity wasincreased by the addition of millimolar concentrationsof Mg 2+ (Zimmermann, 1992). The broad substratespecificity for nucleoside monophosphates of ecto-5 0 -nucleotidase was also observed, with a preference forAMP.  K  M  values vary between cell type and prepara-tion; generally they are in the lower micromolar range(Zimmermann, 1996). The influence of contaminatingenzymes on AMP hydrolysis was discarded, since1.0 mM levamisole or tetramisole did not affect theAMP hydrolysis. The possibility of a pyrophosphatasewas excluded, since no significant enzyme activity wasobserved when 1.0 mM pyrophosphate (Ppi) was usedas substrate instead AMP.Extracellular nucleotides can be hydrolyzed by avariety of enzymes that are located on the cell surface ormay also be soluble in the interstitial medium(Zimmermann, 2001). Extracellular ATP can be hydro-lyzed by NTPDase1, forming AMP, being the 5 0 -nucleo-tidase the final step of the enzymatic chain.Theecto-5 0 -nucleotidaseispresentinvariousanimalstissues, but not on all cell types. Seven human 5 0 -nucleotidaseswithdifferentsubcellularlocalizationhavebeen cloned (Bianchi and Spychala, 2003). This enzymeplays an important role in the formation of adenosinefromextracellularAMPandthesubsequentactivationof P1adenosinereceptors(Zimmermann,2001).Adenosineinduces vasodilatation and inhibition of the immune andinflammatoryresponse(Cronsteinetal.,1992;HaskoandCronstein, 2004). In addition, trichomonads lack theability to synthesize purines and pyrimidines  de novo .Consequently, the salvage pathways to generate nucleo-tides are essential for parasite survival (Heyworth et al.,1982, 1984). Munagala and Wang (2003) demonstrated incorporation of external adenine and guanine into thepurine nucleotides of   T. vaginalis . The purine salvagesystemin T.vaginalis consistsofasinglepathwayoftwoenzymes: PNP (purine nucleoside phosphorylase) andPNK (purine nucleoside kinase). PNP catalyses inter-conversion between purine bases and purine nucleosidesand PNK converts the nucleosides to nucleotides(Heyworth et al., 1982; Miller and Lindstead, 1983).The use of formycin A, a specific inhibitor of bacterialPNP, inhibited  T. vaginalis invitro  growth. Furthermore,lysed epithelial cells during infection release a broadconcentration of purine nucleotides (90% are adeninenucleotides) (Mandel, 1964). Adenosine is the primaryprecursor of the entire purine nucleotide pool in  T.vaginalis andadenosinedeaminase,IMPdehydrogenase,and GMP synthetase activities were identified in theparasite lysate, suggesting a pathway capable of converting adenine to GMP via adenosine (Munagalaand Wang, 2003). Considering the close phylogenybetween  T. vaginalis  and  T. gallinae , it can be suggestedthat the hydrolysis of adenine nucleotides to nucleosideadenosine by a 5 0 -nucleotidase provides the primaryprecursor of the purine nucleotides in the parasites,cooperating to survival and success parasitism.Inaddition,the enzymaticchainpresentin T.gallinae may contribute to escape mechanisms of the parasite bybreaking down ATP and providing adenosine. The 5 0 -nucleotidase activity has been demonstrated in someprotozoan. Tasca et al. (2003) described an ecto-5 0 -nucleotidase in two  T. vaginalis  isolates. These authorshave shown that the enzyme hydrolyzes nucleosidemonophosphates and it is activated by divalent cations,such Mg 2+ and Ca 2+ . The enzyme activities wereinsensitive to levamisole and tetramisole (inhibitors of alkaline phosphatases), whereas AMPCP inhibited theenzymatic activities in both isolates. Furthermore, Tascaet al. (2003) have shown different  K  M  values for bothisolates(111and420 m Mfor30,236and30,238isolates,respectively). In the present report, the ecto-5 0 -nucleo-tidase characterized in intact trophozoites of   T. gallinae wasnotinhibitedbyAMPCP,butpresentedasimilar K  M F.P. Borges et al./Veterinary Parasitology 143 (2007) 106–111  109Table 2Effect of inhibitors on AMP hydrolysis from intact trophozoites of   T.gallinae Inhibitor Concentration(mM)% control enzymeactivityLevamisole 1.0 102.3  7.6Tetramisole 1.0 102  4AMPCP 0.1 106  6Molybdate 1.0 1.6  0.05 * Results were expressed as percentage of control activity (100%).Control 5 0 -nucleotidase activity was 1.83  0.14 nmolPi/min/10 6 tro-phozoites. AMP was used at 3.0 mM, in the presence of MgCl 2 3.0 mM. Data represent the means  S.D. for at least three determi-nations. * Significant difference from control activity (100%) by Student’s  t  test (  p < 0.05).  valuewhencomparedto30,238isolateof  T.vaginalis. Incontrast, the enzyme was strongly inhibited by ammo-niummolybdate,a5 0 -nucleotidaseinhibitor(GottiebandDwyer, 1983; Reilly and Calcutt, 2004; Barros et al.,2000). Gottieb and Dwyer (1983) described a 5 0 -nucleotidase activity in the surface membrane fractionof   Leishmaniadonovani promastigotes.Theenzymewasinhibited by ammonium molybdate, as observed in thepresent findings of this paper. Corte-Real et al. (1993)applied enzyme cytochemistry and immunologicallabeling techniques to characterize a 5 0 -nucleotidase inpromastigote forms of four  Leishmania  species. Ourresultshaveshownanenzymeinintacttrophozoitesof  T.gallinae  that shares kinetic properties with an ecto-5 0 -nucleotidase.The identificationof the physiological significanceof this enzyme could contribute to understanding biochem-ical aspects of   T. gallinae  and mechanisms involved inspecific host–parasite interactions. Adenosine, a pre-cursorofnucleotides intrichomonads,isaproductofthe5 0 -nucleotidase activity. Thus, this enzyme could beconsidered a target for anti-trichomonad drugs in furtherstudies. Acknowledgements Grateful thanks to Patrı´cia de Brum Vieira fortechnical assistance. This work was partially supportedby grants from Conselho Nacional de DesenvolvimentoCientı´fico e Tecnolo´gico (CNPq) and Fundac¸a˜o deAmparo a` Pesquisa do Estado do Rio Grande do Sul(FAPERGS). References Baker, J.R., 1986. Trichomoniasis, a major cause of vomiting inbudgerigars. Vet. Rec. 118, 447–449.Barros, F.S., De Menezes, L.F., Pinheiro, A.A.S., Silva, E.F., Lopes,A.H.C.S., De Souza, W., Meyer-Fernandes, J.R., 2000. Ectonu-cleotide diphosphohydrolase activities in  Entamoeba histolytica .Arch. Biochem. Biophys. 375, 304–314.Berreˆdo-Pinho, M., Peres-Sampaio, C.E., Chrispim, P.P.M., Belmont-Firpo,R.,Lemos,A.P.,Martiny,A.,Vannier-Santos,M.A.,Meyer-Fernandes, J.R., 2001. A Mg-dependent ecto-ATPase in  Leishma-nia amazonensis  and its possible role in adenosine acquisition andvirulence. Arch. Biochem. 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