RESUMEN
We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.
Asunto(s)
Antígenos CD/química , Antígenos CD/inmunología , Antígenos de Protozoos/metabolismo , Apirasa/química , Apirasa/inmunología , Leishmania infantum/enzimología , Leishmania infantum/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD/aislamiento & purificación , Antígenos CD/metabolismo , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Apirasa/aislamiento & purificación , Apirasa/metabolismo , Perros , Leishmania infantum/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.
Asunto(s)
Apirasa/inmunología , Leishmania mexicana/enzimología , Leishmaniasis Cutánea/diagnóstico , Solanum tuberosum/enzimología , Animales , Variación Antigénica , Apirasa/aislamiento & purificación , Apirasa/metabolismo , Western Blotting , Reacciones Cruzadas , Progresión de la Enfermedad , Electroforesis en Gel de Poliacrilamida , Epítopos , Femenino , Isoenzimas/inmunología , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Ratones , Ratones Endogámicos BALB CRESUMEN
The fact that the Schistosoma mansoni egg has two ATP diphosphohydrolase (EC 3.6.1.5) isoforms with different net charges and an identical molecular weight of 63,000, identified by non-denaturing polyacrylamide gel electrophoresis and immunological cross-reactivity with potato apyrase antibodies, is shown. In soluble egg antigen (SEA), only the isoform with the lower net negative charge was detected and seemed to be the predominant species in this preparation. By confocal fluorescence microscopy, using anti-potato apyrase antibodies, the S. mansoni egg ATP diphosphohydrolase was detected on the external surface of miracidium and in von Lichtenberg's envelope. Intense fluorescence was also seen in the outer side of the egg-shell, entrapped by the surface microspines, suggesting that a soluble isoform is secreted. ATP diphosphohydrolase antigenicity was tested using the vegetable protein as antigen. The purified potato apyrase was recognized in Western blots by antibodies present in sera from experimentally S. mansoni-infected mice. In addition, high levels of IgG anti-ATP diphosphohydrolase antibodies were detected by ELISA in the same sera. This work represents the first demonstration of antigenic properties of S. mansoni ATP diphosphohydrolase and immunological cross-reactivity between potato apyrase and sera from infected individuals.
Asunto(s)
Antígenos Helmínticos/química , Apirasa/química , Schistosoma mansoni/enzimología , Animales , Antígenos Helmínticos/aislamiento & purificación , Antígenos Helmínticos/metabolismo , Apirasa/inmunología , Apirasa/aislamiento & purificación , Apirasa/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Isoenzimas , Hígado/parasitología , Ratones , Microscopía Fluorescente , Peso Molecular , Schistosoma mansoni/inmunología , Schistosoma mansoni/metabolismoRESUMEN
In the present work we characterized the ecto-ATP diphosphohydrolase activity of the trypanosomatid parasite Herpetomonas muscarum muscarum. This parasite hydrolyzed ATP at a rate of 15.52 nmol Pi/mg protein/min and this activity reached a maximum at pH 7.5. Classical inhibitors of acid phosphatases, such as sodium orthovanadate (NaVO3), sodium fluoride (NaF), and ammonium molybdate presented no effect on this activity. MgCl2, ZnCl2, and MnCl2 stimulated the ATP hydrolysis by H. m. muscarum. The ecto-ATPase activity was insensitive to oligomycin and sodium azide, two inhibitors of mitochondrial Mg-ATPase, bafilomycin A1, a V-ATPase inhibitor, ouabain, a Na(+)+K+-ATPase inhibitor and to levamizole, an inhibitor of alkaline phosphatase. An extracellular impermeant inhibitor 4,4'-diisothiocyanostylbene 2',2'-disulfonic acid (DIDS) and a inhibitor of some ecto-ATPases, suramin, which is also a competitive antagonist of P2-purinergic receptors, promoted a great inhibition on the ATP hydrolysis. This enzyme is able to hydrolysis ATP, ADP, UTP, and UDP, but not GTP, GDP, CTP, or CDP. ADP inhibited the enzymatic activity in a concentration dependent manner, reaching 70% inhibition.
Asunto(s)
Apirasa/aislamiento & purificación , Apirasa/metabolismo , Trypanosomatina/enzimología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Antígenos CD , Cationes Bivalentes/farmacología , Activadores de Enzimas/análisis , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas/efectos de los fármacos , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Especificidad por Sustrato , Suramina/farmacología , Tripanocidas/farmacologíaRESUMEN
An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.
Asunto(s)
Apirasa/metabolismo , Leishmania/enzimología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apirasa/antagonistas & inhibidores , Apirasa/aislamiento & purificación , Calcio/química , Membrana Celular/enzimología , Membrana Celular/ultraestructura , Inhibidores Enzimáticos/farmacología , Femenino , Concentración de Iones de Hidrógeno , Leishmania/ultraestructura , Levamisol/farmacología , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Molibdeno/química , Azida Sódica/química , Especificidad por SustratoRESUMEN
Este trabalho mostrou a purificação de uma apirase em extratos de tegumentos de vermes adultos de S. mansoni com razão de hidrólise de ADP/ATP aproximadamente 2. Análise da amostra por MALDI-TOF evidenciou a presença de helicase e carboxilesterase, sendo que a atividade de hidrólise de ADP ainda não foi descrita para estas proteínas. Através de ®Western blot¼ mostrou-se que a proteína purificada não tem epítopos reconhecíveis por anticorpo anti-apirase de batata, e que a imunoreatividade cruzada com apirase da família CD39 está presente na fração insolúvel que não foi utilizada no processo de purificação. Esta tese caracteriza também a utilização de ®primers¼ consenso degenerados na construção de bibliotecas de cDNA com PCR de baixa estringência...
Asunto(s)
Animales , Ratones , Antígenos/uso terapéutico , Apirasa/aislamiento & purificación , Clonación de Organismos , Biblioteca de Genes , Schistosoma mansoni/inmunología , Esquistosomiasis/prevención & control , Western Blotting , Electroforesis en Gel de Poliacrilamida , Reacción en Cadena de la PolimerasaRESUMEN
Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.
Asunto(s)
Adenosina Difosfato/metabolismo , Apirasa/metabolismo , Nucleótidos/metabolismo , Proteínas de Plantas/metabolismo , Solanum tuberosum/enzimología , Apirasa/química , Apirasa/aislamiento & purificación , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Solanum tuberosum/química , Espectrometría de FluorescenciaRESUMEN
Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.
Asunto(s)
Adenosina Difosfato/metabolismo , Apirasa/metabolismo , Nucleótidos/metabolismo , Solanum tuberosum/enzimología , Apirasa/química , Apirasa/aislamiento & purificación , Cesio/química , Cesio/metabolismo , Yodo/química , Yodo/metabolismo , Isoenzimas/química , Solanum tuberosum/química , Espectrometría de FluorescenciaRESUMEN
ATP diphosphohydrolases are described as ecto-enzymes in several tissues. In the present study, synaptic plasma membrane (SPM) was exposed to a series of agents used to distinguish between peripheral (hydrophilic), G-PI-anchored and transmembrane-polypeptide-anchored membrane proteins. These procedures included: (a) nondetergent extraction, (b) Triton X-114 phase partitioning, (c) phosphatidylinositol-specific phospholipase C (PI-PLC) extraction and (d) protease incubation. In cases (a), (c) and (d) the SPM was incubated with different agents and the ATPase-ADPase activities and the protein concentration was determined in the original sample, in the pellet and in the supernatant obtained after 100,000 g centrifugation. In procedure (b), the SPM was solubilized in 1% triton X-114 and submitted to phase separation onto a sucrose cushion. The aqueous and detergent rich phases obtained by this treatment were assayed for ATPase-ADPase activities and protein determination. The results obtained suggest an intrinsic behaviour for ATP diphosphohydrolase since none of the nondetergent treatments was efficient in removing the enzyme from SPM. Moreover, ATPase and ADPase activities were recovered predominantly (> 50%) in the detergent-rich phase obtained by Triton X-114 partitioning. The enzyme was not released by PI-PLC or proteases. These results indicate that the enzyme is not a GPI-anchored protein, but is probably deeply anchored on the plasma membrane in agreement with the amino acid sequence of the enzyme recently published.
Asunto(s)
Apirasa/aislamiento & purificación , Encéfalo/enzimología , Proteínas de la Membrana/aislamiento & purificación , Membranas Sinápticas/enzimología , Animales , Apirasa/metabolismo , Detergentes , Masculino , Proteínas de la Membrana/metabolismo , Octoxinol , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Polietilenglicoles , Ratas , Ratas Wistar , Solubilidad , Fosfolipasas de Tipo C/metabolismoRESUMEN
ATP diphosphohydrolase from tegumental membranes of Schistosoma mansoni was solubilized with Triton X-100 plus deoxycholate and separated by preparative nondenaturing polyacrylamide gel electrophoresis. Two isoforms with ATP-hydrolytic activity were identified and excised from nondenaturing gels. For each of the active bands, two protein bands (63 and 55 kDa) were detected with Coomassie Blue staining, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots developed with polyclonal anti-potato apyrase antibody revealed a single protein of 63 kDa, either with samples excised from active bands or with total S. mansoni tegument. Anti-potato apyrase antibody immobilized on Sepharose-Protein A depleted over 95% of ATPase and ADPase activities from detergent-solubilized tegument. Confocal laser scanning microscopy showed anti-potato apyrase antibody on the outer surface of S. mansoni tegument. A different antibody against a fusion protein derived from recently cloned Toxoplasma gondii nucleoside triphosphate hydrolase (Bermudes, D., Peck, K. R., Afifi, M. A., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260) revealed the same 63-kDa band in Western blots of S. mansoni tegument. Since anti-potato apyrase antibodies exhibited cross-reactivity with S. mansoni ATP diphosphohydrolase, we decided to gain further information on the primary structure of potato apyrase by sequencing the protein. Three novel peptides were obtained: amino-terminal sequence and two internal sequences from tryptic fragments. Eight sequences recently deposited in the data bank, including that of T. gondii nucleoside triphosphate hydrolase, have considerable homologies to potato apyrase suggesting a new family of nucleoside triphosphatases which contains a conserved motif (I/V)(V/M/I)(I/L/F/C)DAGS(S/T) near the amino-terminal. Antibody cross-reactivities in the present work suggest that conserved epitopes from S. mansoni ATP diphosphohydrolase are present in this family of nucleotide-splitting enzymes.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Apirasa/aislamiento & purificación , Apirasa/metabolismo , Schistosoma mansoni/enzimología , Toxoplasma/enzimología , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Nucleósido-Trifosfatasa , Homología de Secuencia de Aminoácido , Solanum tuberosum/enzimologíaRESUMEN
This review aimed at discussing the protocols used to characterize and to distinguish ATP diphosphohydrolases from other enzymes which can promote the degradation of ATP and ADP, since there is a confusion about the identily of this enzyme and ATPases.