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José Meyer-fernandes - One of the best experts on this subject based on the ideXlab platform.
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Ecto-nucleotidase activities in the fat body of Rhodnius prolixus.
Archives of Insect Biochemistry and Physiology, 2020Co-Authors: Peter F Entringer, Katia C Gondim, José Meyer-fernandesAbstract:In this study, we describe the ability of intact fat body of an insect, Rhodnius prolixus, to hydrolyze extracellular ATP. In these fat bodies, the ATP hydrolysis was low in the absence of any divalent metal, and was stimulated by MgCl2. Both activities (in the absence or presence of MgCl2) were linear with time for at least 30 min. In order to confirm the observed nucleotidase activities as ecto-nucleotidases, we used an impermeant inhibitor, DIDS (4, 4′-diisothiocyanostylbene 2′-2′-disulfonic acid). This reagent inhibited both nucleotidase activities and its inhibitory effect was suppressed by ATP. Both ecto-nucleotidase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin, ouabain, vanadate, molybdate, sodium fluoride, levamizole, tartrate, p-NPP, sodium phosphate, and suramin. Concanavalin A, activator of some Ecto-ATPases, was able to stimulate the Mg2+-independent nucleotidase activity, but not the Mg2+-dependent one. The Mg2+-independent nucleotidase activity was enhanced with increases in the pH in the range between 6.4–8.0, but the Mg2+-dependent nucleotidase activity was not affected. Besides MgCl2 , the ecto-ATPase activity was also stimulated by CaCl2, MnCl2, and SrCl2, but not by ZnCl2. ATP, ADP, and AMP were the best substrates for the Mg2+-dependent ecto-nucleotidase activity, and CTP, GTP, and UTP produced very low reaction rates. However, the Mg2+-independent nucleotidase activity recognized all these nucleotides producing similar reaction rates, but GTP was a less efficient substrate. The possible role of the two ecto-nucleotidase activities present on the cell surface of fat body of Rhodnius prolixus, which are distinguished by their substrate specificity and their response to Mg2+, is discussed. Arch. Insect Biochem. Physiol. 61:1–9, 2006. © 2005 Wiley-Liss, Inc.
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Inhibition of ecto-ATPase activities impairs HIV-1 infection of macrophages
Immunobiology, 2014Co-Authors: Julieta Schachter, Kelly Valcárcel Delgado, Victor Barreto-de-souza, Dumith Chequer Bou-habib, Pedro M. Persechini, José Meyer-fernandesAbstract:Nucleotides and nucleosides are secreted into extracellular media at different concentrations as a consequence of different physiologic and pathological conditions. Ecto-nucleotidases, enzymes present on the surface of most cells, hydrolyze these extracellular nucleotides and reduce the concentration of them, thus affecting the activation of different nucleotide and nucleoside receptors. Also, ecto-nucleotidases are present in a number of microorganisms and play important roles in host-pathogen interactions. Here, we characterized the ecto-ATPase activities present on the surface of HIV-1 particle and human macrophages as well. We found that the kinetic properties of HIV-1 and macrophage Ecto-ATPases are similar, suggesting that the enzyme is the same. This ecto-ATPase activity was increased in macrophages infected in vitro with HIV-1. Using three different non-related ecto-ATPase inhibitors-POM-1, ARL67156 and BG0-we showed that the inhibition of these macrophage and viral ecto-ATPase activities impairs HIV-1 infection. In addition, we also found that elevated extracellular concentrations of ATP inhibit HIV-1 production by infected macrophages.
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Leishmania amazonensis: Increase in ecto-ATPase activity and parasite burden of vinblastine-resistant protozoa.
Experimental Parasitology, 2014Co-Authors: Naira Lígia Lima Giarola, Thaís S. Silveira, Job D. F. Inacio, Lisvane Paes Vieira, Elmo E. Almeida-amaral, José Meyer-fernandesAbstract: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 L. 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.
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Trypanosoma cruzi: effects of heat shock on ecto-ATPase activity.
Experimental Parasitology, 2013Co-Authors: Naira Lígia Lima Giarola, Katia C Gondim, Elmo Eduardo De Almeida-amaral, Itallo Collopy-júnior, André L. Fonseca-de-souza, David Majerowicz, Lisvane Silva Paes, José Meyer-fernandesAbstract:In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.
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CrATP as a new inhibitor of Ecto-ATPases of trypanosomatids.
Parasitology, 2009Co-Authors: Otacilio C. Moreira, José Meyer-fernandes, P. F. Rios, F. F. Esteves, Hector BarrabinAbstract:Trypanosomatid protozoa include heteroxenic species some of them pathogenic for men, animals and plants. Parasite membrane contains ecto-enzymes whose active sites face the external medium rather than the cytoplasm. Herpetomonas sp. displayed a Mg2+-dependent ecto-ATPase activity, a Mg-independent ecto-ADPase and an ecto-phosphatase activity. Both, the ecto-ADPase and phosphatase activities were insensitive to CrATP (chromium(III) adenosine 5'-triphosphate complex). Ecto-ATPase activity was reversibly inhibited. At 2 mm ATP the apparent Ki was 4 x 7+/-1 x 0 microm but a fraction of about 40-50% was insensitive to CrATP. Remarkably, at low substrate concentration (0 x 2 mm) more than 90% of the ecto-ATPase was inhibited with Ki=0 x 33+/-0 x 10 microm. These parameter dependences are interpreted as the presence of 2 Ecto-ATPases activities, one of them with high ATP apparent affinity and sensitivity to CrATP. DIDS (4,4 diisothiocyanatostilbene 2,2' disulfonic acid), suramin and ADP were also effective as inhibitors. Only ADP presented no additive inhibition with CrATP. The pattern of partial inhibition by CrATP was also observed for the ecto-ATPase activities of Leishmania amazonensis, Trypanosoma cruzi and Trypanosoma rangeli. CrATP emerges as a new inhibitor of Ecto-ATPases and as a tool for a better understanding of properties and role of Ecto-ATPases in the biology of parasites.
Terence L. Kirley - One of the best experts on this subject based on the ideXlab platform.
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Expression and characterization of human ecto-ATPase and chimeras with CD39 ecto-apyrase.
Iubmb Life, 2000Co-Authors: Carrie A. Hicks-berger, Terence L. KirleyAbstract:Summary Human ecto-ATPase (ecto-nucleoside triphosphate diphosphohydrolase 2 [eNTPDase2], also known as CD39L1) has been expressed and characterized in COS cells. It exhibits some unusual enzymologythatissimilartoafewmembersofthisclassofproteins but different from the majority of the family members. Hydrolysis of ATP by human ecto-ATPase is nonlinear with time, and its activity is stimulated/stabilized by both the lectin concanavalin A and the chemical cross-linking agent disuccinimidylsuberate. Like other members of the eNTPDase family, the human ecto-ATPase is a tetramer, the activity of which depends on its glycosylation. Chimeras of this protein with human CD39 (eNTPDase1) were constructed to test the hypothesis that the N-terminal half of these proteins regulates nucleotide specie city. The two chimeras generated demonstrated that the N-terminal half of these proteins is crucial for determining the relative activities of the nucleoside diand triphosphatases. Chemical cross-linking of the two chimeras suggeststhatdisuccinimidylsuberateinteractswith theC-terminal half of ecto-ATPase in a manner that results in an increase of activity for both the ecto-ATPase and the ecto-apyrase/ecto-ATPase chimera. IUBMBLife, 50: 43‐50, 2000
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Expression and characterization of chicken muscle ecto-ATPase in mammalian COS cells.
Iubmb Life, 1999Co-Authors: Terence L. Kirley, Lamar K. Gerber, Thomas M. SmithAbstract:Chicken muscle ecto-ATPase has unusual enzyme kinetics and properties not found in many other E-type ATPases. To determine whether the unique properties of the chicken ecto-ATPase are inherent in the protein sequence and not mediated by some unique property of the chicken system, we have spliced together two partial cDNAs encoding the ecto-ATPase. The enzymatic properties of the COS (green monkey kidney) cell-expressed protein are indistinguishable from the purified chicken gizzard ecto-ATPase, including a 2- to 3-fold stimulation of membrane-bound activity by crosslinking and lectins, properties not shared by most other E-type ATPases. The expressed enzyme is specific for nucleotide triphosphates (ATPase:ADPase hydrolysis ratio of 26:1) and is inhibited by Cibacron Blue (IC50 = 10 microM). The active, expressed enzyme can be affinity-purified with Cibacron Blue, is relatively resistant to deglycosylation, and is less stable than other E-type ATPases. Expression in the presence of tunicamycin resulted in an inactive, unfolded enzyme.
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Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the Ecto-ATPases and CD39 ecto-apyrases
Biochimica et Biophysica Acta, 1998Co-Authors: Thomas M. Smith, Terence L. KirleyAbstract:Abstract An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-apyrase. However, this hydrolysis ratio is intermediate between that observed for the Ecto-ATPases and the CD39 ecto-apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141–214). Quantitative analyses of amino acid identities and similarities between this ecto-apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the Ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both Ecto-ATPases and CD39 ecto-apyrases are derived.
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Immunological detection of ECTO‐atpase in chicken and rat tissues: Characterization, distribution, and a cautionary note
Iubmb Life, 1998Co-Authors: Thomas M. Smith, Stephanie Ann Lewis Carl, Terence L. KirleyAbstract:We have generated a polyclonal antibody (CKG2) against native chicken gizzard ecto-ATPase for immunolocalization and immunoprecipitation. Active ecto-ATPase is immunoprecipitated from solubilized chicken and rat membranes and shown to be localized to the plasma membrane of the chicken smooth muscle cells. This antibody is specific for the Ecto-ATPases, since the more abundant chicken stomach ecto-apyrase is not recognized in immunoprecipitation, western blot or immunolocalization analyses. The CKG2 antibody cross-reacts with mammalian (rat) ecto-ATPase in western blots, with testis being the most abundant source. Interestingly, when the same rat membranes are analyzed by western blot under non-reducing conditions, the 66 kDa ecto-ATPase is not recognized, instead a 200 kDa protein is detected, previously postulated to be an oligomer of ecto-ATPase. However, this 200 kDa cross-reacting protein is not related to the Ecto-ATPases, but is instead an immunoglobulin binding protein, comprised of 50 kDa subunits.
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Purification, Characterization, and Molecular Cloning of the Chicken Gizzard Smooth Muscle Ecto-ATPase
Ecto-ATPases, 1997Co-Authors: Terence L. Kirley, James G. StoutAbstract:The ecto-ATPase from chicken smooth muscle was solubilized, purified, and characterized. Mono- and polyclonal antibodies were raised and the tissue distribution based on Western analysis was determined. N-terminal and internal protein sequences were determined and used to design degenerate oligonucleotide probes to screen a chicken muscle cDNA library. Two overlapping partial clones encoding most of the ecto-ATPase were isolated and sequenced. A unified theory as to the mechanism by which many varied types of molecules modulate ecto-ATPase activity was developed, and the theory was supported by cross-linking data.
Aileen F. Knowles - One of the best experts on this subject based on the ideXlab platform.
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The GDA1_CD39 superfamily: NTPDases with diverse functions
Purinergic Signalling, 2011Co-Authors: Aileen F. KnowlesAbstract:The first comprehensive review of the ubiquitous “Ecto-ATPases” by Plesner was published in 1995. A year later, a lymphoid cell activation antigen, CD39, that had been cloned previously, was shown to be an ecto-ATPase. A family of proteins, related to CD39 and a yeast GDPase, all containing the canonical apyrase conserved regions in their polypeptides, soon started to expand. They are now recognized as members of the GDA1_CD39 protein family. Because proteins in this family hydrolyze nucleoside triphosphates and diphosphates, a unifying nomenclature, nucleoside triphosphate diphopshohydrolases (NTPDases), was established in 2000. Membrane-bound NTPDases are either located on the cell surface or membranes of intracellular organelles. Soluble NTPDases exist in the cytosol and may be secreted. In the last 15 years, molecular cloning and functional expression have facilitated biochemical characterization of NTPDases of many organisms, culminating in the recent structural determination of the ecto-domain of a mammalian cell surface NTPDase and a bacterial NTPDase. The first goal of this review is to summarize the biochemical, mutagenesis, and structural studies of the NTPDases. Because of their ability in hydrolyzing extracellular nucleotides, the mammalian cell surface NTPDases (the ecto-NTPDases) which regulate purinergic signaling have received the most attention. Less appreciated are the functions of intracellular NTPDases and NTPDases of other organisms, e.g., bacteria, parasites, Drosophila, plants, etc. The second goal of this review is to summarize recent findings which demonstrate the involvement of the NTPDases in multiple and diverse physiological processes: pathogen-host interaction, plant growth, eukaryote cell protein and lipid glycosylation, eye development, and oncogenesis.
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Enzymatic and transcriptional regulation of human ecto-ATPase/E-NTPDase 2.
Archives of Biochemistry and Biophysics, 2003Co-Authors: Aileen F. Knowles, Wei-chieh ChiangAbstract:We have characterized the regulation of expressed human ecto-ATPase (E-NTPDase 2), a cell surface integral membrane glycoprotein. Ecto-ATPase activity is inhibited by parameters that decrease membrane protein interaction, i.e., detergents and high temperatures. These inhibitory effects are overcome when membranes are pretreated with concanavalin A or chemical cross-linking agents that increase the amounts of ecto-ATPase oligomers. Cross-linking agents also abrogate substrate inactivation of the ecto-ATPase, a unique characteristic of the enzyme. These effects indicate that the magnitude of negative substrate regulation is dependent on quaternary structures of the protein, which likely involves interaction of transmembrane domains. The importance of transmembrane domains of ecto-ATPase in activity modulation is demonstrated further by the stimulatory effect of digitonin, a steroid glycoside that preferentially interacts with cholesterol in the membranes but does not promote oligomer formation. These results indicate that ecto-ATPase activity is regulated by a multitude of mechanisms, some of which may have physiological significance. Ecto-ATPase is also susceptible to transcriptional regulation. Ecto-ATPase gene expression is increased in a human hepatoma whereas it is undetectable in the normal liver.
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Regulation of chicken gizzard ecto-ATPase activity by modulators that affect its oligomerization status.
Archives of Biochemistry and Biophysics, 2001Co-Authors: Charles C. Caldwell, Stephen C. Hornyak, Erik Pendleton, Dawn Campbell, Aileen F. KnowlesAbstract:The major ectonucleoside triphosphate phosphohydrolase in the chicken gizzard smooth muscle membranes is an ecto-ATPase, an integral membrane glycoprotein belonging to the E-ATPase (or E-NTPDase) family. The gizzard ecto-ATPase is distinguished by its unusual kinetic properties, temperature dependence, and response to a variety of modulators. Compounds that promote oligomerization of the enzyme protein, i.e., concanavalin A, chemical cross-linking agent, and eosin iodoacetamide, increase its activity. Compounds that inhibit some ion-motive ATPases, e.g., sulfhydryl reagents, xanthene derivatives, NBD-halides, and suramin, also inhibit the gizzard ecto-ATPase, but not another E-ATPase, the chicken liver ecto-ATP-diphosphohydrolase, which contains the same conserved regions as the ecto-ATPase. Furthermore, inhibition of the gizzard ecto-ATPase by these compounds as well as detergents is not prevented by preincubation of the membranes with the substrate, ATP, indicating that their interaction with the enzyme occurs at a locus other than the catalytic site. On the other hand, the inhibitory effect of these compounds, except suramin, is abolished or reduced if the membranes are preincubated with concanavalin A. It is concluded that these structurally unrelated modulators exert their effect by interfering with the oligomerization of the ecto-ATPase protein. Our findings suggest that, under physiological conditions, the gizzard smooth muscle ecto-ATPase may exhibit a range of activities determined by membrane events that affect the status of oligomerization of the enzyme.
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Is the Rat Liver Ecto-ATPase Identical to C-CAM/PP120, a Cell Adhesion Molecule?
Ecto-ATPases, 1997Co-Authors: Aileen F. Knowles, Sandra L. MurrayAbstract:The publication of a report on the molecular cloning of the rat liver ecto-ATPase1 in 1989 was considered a major break-through in ecto-ATPase research, especially since no other ecto-ATPase had been purified at that time. The extensive homology of the cDNA sequence of the rat liver ecto-ATPase with human biliary glycoprotein I (BGPI) also gave hope that the function of the Ecto-ATPases might soon be revealed since functional studies of BGPI and related proteins in the carcinoembryonic antigen (CEA) gene family had begun and a cell adhesion function had been suggested2, 3. Subsequent reports on amino acid sequence similarity of the rat liver ecto-ATPase with a rat liver cell adhesion molecule (cell-CAM 105)4, cross-reactivity of the ecto-ATPase and eell-CAM105 with antibodies generated against the other protein5, and functional assays4–6 unambiguously established that the BGP-like cDNA codes for a cell adhesion molecule. However, the important question of the relationship of ATPase activity and cell adhesion function was not addressed. In later reports where consequences of manipulating the cDNA on cell aggregation were described6–8, there was no concomitant evaluation of the ATPase activity of the mutants.
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Human Tumor Ecto-ATPases
Ecto-ATPases, 1997Co-Authors: Aileen F. KnowlesAbstract:When normal cells are transformed into tumor cells, numerous alterations occur on the cell membrane. These include increased transport of nutrients, changes of protein and lipid composition, greater agglutinability by lectins, decreased intercellular cell adhesion, and altered activities of receptors and cell surface enzymes1, 2. Studies of a plasma membrane ATPase in normal and tumor tissues began nearly forty years ago, first by cytochemical staining3 and later by biochemical determination4. While the ATPase activity was compromised by the necessary use of glutaraldehyde and lead in the cytochemical staining procedure, the method was widely used in localizing and quantifying the cell-surface ATPase, i.e. ecto-ATPase.
Jose Roberto Meyerfernandes - One of the best experts on this subject based on the ideXlab platform.
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trypanosoma rangeli characterization of a mg dependent ecto atp diphosphohydrolase activity
Experimental Parasitology, 2006Co-Authors: Fabio V Fonseca, Andre Luiz Fonseca De Souza, Ana C Mariano, Peter F Entringer, Katia C Gondim, Jose Roberto MeyerfernandesAbstract: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 × 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 × 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.
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characterization of an ecto atpase of tritrichomonas foetus
Veterinary Parasitology, 2002Co-Authors: J B Jesus, Angela H.c.s. Lopes, Jose Roberto MeyerfernandesAbstract:Abstract In this work, we describe the ability of living Tritrichomonas foetus to hydrolyze extracellular ATP. The addition of MgCl2 to the assay medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half maximal stimulation of ATP hydrolysis was obtained with 0.46 mM MgCl2. The ecto-ATPase activity was also stimulated by MnCl2 and CaCl2, but not by SrCl2. The Mg2+-dependent ATPase presents two apparent Km values for Mg-ATP2− (Km1=0.03 mM and Km2=2.01 mM). ATP was the best substrate for this enzyme, although other nucleotides such as ITP, CTP, UTP also produced high reaction rates. GTP produced a low reaction rate and ADP was not a substrate for this enzyme. The Mg2+-dependent 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 and levamizole. The acid phosphatase inhibitors (vanadate and molybdate) inhibited about 60–70% of the Mg2+-independent ecto-ATPase activity, suggesting that the ATP hydrolysis measured in the absence of any metal divalent could, at least in part, also be catalyzed by an ecto-phosphatase present in this cell. In order to confirm the observed Mg2+-dependent activity as an ecto-ATPase, we used an impermeant inhibitor, 4,4′-diisothiocyanostylbene-2′,2′-disulfonic acid (DIDS) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some Ecto-ATPases. These two reagents inhibited the Mg2+-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase was stimulated by more than 90% by 50 mM d -galactose. Since previous results showed that d -galactose exposed on the surface of host cells is involved with T. foetus adhesion, the Mg2+-dependent ecto-ATPase may be involved with cellular adhesion and possible pathogenicity.
Thomas M. Smith - One of the best experts on this subject based on the ideXlab platform.
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Expression and characterization of chicken muscle ecto-ATPase in mammalian COS cells.
Iubmb Life, 1999Co-Authors: Terence L. Kirley, Lamar K. Gerber, Thomas M. SmithAbstract:Chicken muscle ecto-ATPase has unusual enzyme kinetics and properties not found in many other E-type ATPases. To determine whether the unique properties of the chicken ecto-ATPase are inherent in the protein sequence and not mediated by some unique property of the chicken system, we have spliced together two partial cDNAs encoding the ecto-ATPase. The enzymatic properties of the COS (green monkey kidney) cell-expressed protein are indistinguishable from the purified chicken gizzard ecto-ATPase, including a 2- to 3-fold stimulation of membrane-bound activity by crosslinking and lectins, properties not shared by most other E-type ATPases. The expressed enzyme is specific for nucleotide triphosphates (ATPase:ADPase hydrolysis ratio of 26:1) and is inhibited by Cibacron Blue (IC50 = 10 microM). The active, expressed enzyme can be affinity-purified with Cibacron Blue, is relatively resistant to deglycosylation, and is less stable than other E-type ATPases. Expression in the presence of tunicamycin resulted in an inactive, unfolded enzyme.
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Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the Ecto-ATPases and CD39 ecto-apyrases
Biochimica et Biophysica Acta, 1998Co-Authors: Thomas M. Smith, Terence L. KirleyAbstract:Abstract An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-apyrase. However, this hydrolysis ratio is intermediate between that observed for the Ecto-ATPases and the CD39 ecto-apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141–214). Quantitative analyses of amino acid identities and similarities between this ecto-apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the Ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both Ecto-ATPases and CD39 ecto-apyrases are derived.
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Immunological detection of ECTO‐atpase in chicken and rat tissues: Characterization, distribution, and a cautionary note
Iubmb Life, 1998Co-Authors: Thomas M. Smith, Stephanie Ann Lewis Carl, Terence L. KirleyAbstract:We have generated a polyclonal antibody (CKG2) against native chicken gizzard ecto-ATPase for immunolocalization and immunoprecipitation. Active ecto-ATPase is immunoprecipitated from solubilized chicken and rat membranes and shown to be localized to the plasma membrane of the chicken smooth muscle cells. This antibody is specific for the Ecto-ATPases, since the more abundant chicken stomach ecto-apyrase is not recognized in immunoprecipitation, western blot or immunolocalization analyses. The CKG2 antibody cross-reacts with mammalian (rat) ecto-ATPase in western blots, with testis being the most abundant source. Interestingly, when the same rat membranes are analyzed by western blot under non-reducing conditions, the 66 kDa ecto-ATPase is not recognized, instead a 200 kDa protein is detected, previously postulated to be an oligomer of ecto-ATPase. However, this 200 kDa cross-reacting protein is not related to the Ecto-ATPases, but is instead an immunoglobulin binding protein, comprised of 50 kDa subunits.
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Ecto-ATPase of Tetrahymena
Ecto-ATPases, 1997Co-Authors: Thomas M. Smith, Terence L. Kirley, Todd M. HennesseyAbstract:An E-type ecto-ATPase activity was demonstrated in the unicellular eukaryote, Tetrahymena. The in vivo membrane bound activity was increased 4 fold by exposing cells to 50mM NaCl for 1 hour, suggesting its activity can be regulated. It is also released in a soluble form over time. Both forms are recognized by antibodies to the smooth muscle ecto-ATPase of chicken, all with molecular weights of about 66kD. The soluble form was purified over 660 fold and it showed a single 66kD band on SDS-PAGE. This ecto-ATPase only hydrolyzes nucleoside triphosphates but it prefers ATP over GTP by 4 fold. In purinergic reception, GTP is normally more effective as a chemorepellent in these cells than ATP. Their sensitivities were greatly increased by inhibiting nTP hydrolysis. Cells were most sensitive to the non-hydrolyzable analogs (beta-gamma methylene GTP and ATP) at similar concentrations. This suggests that the ecto-ATPase may play a role in sensitivity and selectivity of the purinergic responses of Tetrahymena.
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A soluble ecto-ATPase from Tetrahymena thermophila: Purification and similarity to the membrane-bound ecto-ATPase of smooth muscle
Scopus, 1997Co-Authors: Thomas M. Smith, Terence L. Kirley, Todd M. HennesseyAbstract:Abstract 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 Ca 2+ or Mg 2+ 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 ectoATPase 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.
