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1
Darvish, A., R. W. Pomerantz, P. G. Zografides, and P. J. Metting. "Contribution of cytosolic and membrane-bound 5'-nucleotidases to cardiac adenosine production." American Journal of Physiology-Heart and Circulatory Physiology 271, no. 5 (November 1, 1996): H2162—H2167. http://dx.doi.org/10.1152/ajpheart.1996.271.5.h2162.
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The purpose of this study was to evaluate the relative contributions of AMP-specific cytosolic 5'-nucleotidase and ecto-5'-nucleotidase to cardiac adenosine production and its regulation by ADP and Mg2+. 5'-Nucleotidase activity was measured spectrophotometrically in the total homogenate, the 150,000-g supernatant fraction (cytosolic 5'-nucleotidase), and the membrane pellet fraction (ecto-5'-nucleotidase) of dog left ventricles. Increasing [MgCl2] over a range from 0 to 6 mmol/l increased 5'-nucleotidase activity in both the supernatant and pellet; only cytosolic 5'-nucleotidase exhibited an absolute requirement for Mg2+. ADP, (20-480 mumol/l) activated supernatant and inhibited membrane-bound 5'-nucleotidase activity. At 80 mumol/l ADP, 5 mmol/l MgCl2, 100 mumol/l AMP, and pH 7.3, the average 5'-nucleotidase activities of the supernatant vs. pellet were 74% of total and 26% of total, respectively. Total adenosine production in unfractionated samples of ventricular homogenates decreased an average of 73% by specific inhibition of cytosolic 5'-nucleotidase, using antibodies against the cytosolic enzyme, and 46% by specific inhibition of ecto-5'-nucleotidase with alpha, beta-methylene adenosine 5'-diphosphate (AOPCP). These findings support the hypotheses that 1) both cytosolic and ecto-5'-nucleotidase contribute to cardiac adenosine production in dog heart homogenates; 2) AMP-specific cytosolic 5'-nucleotidase activity exceeds ecto-5'-nucleotidase activity at physiological concentrations of ADP, AMP, and Mg2+; and 3) Mg2+ is an important regulator of cardiac adenosine production via activation of both ecto- and AMP-specific cytosolic 5'-nucleotidases.
2
Stochaj, U., and H. G. Mannherz. "Affinity labelling of 5′-nucleotidases with 5′-p-fluorosulphonylbenzoyladenosine." Biochemical Journal 266, no. 2 (March 1, 1990): 447–51. http://dx.doi.org/10.1042/bj2660447.
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5′-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). 5′-FSBA irreversibly inactivates 5′-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5′-nucleotidase, abolished the inactivation by 5′-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5′-FSBA, which was employed for the radiolabelling of chicken gizzard 5′-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5′-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5′-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5′-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5′-nucleotidase by 5′-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.
3
Headrick, J. P., and R. J. Willis. "5′-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart." Biochemical Journal 261, no. 2 (July 15, 1989): 541–50. http://dx.doi.org/10.1042/bj2610541.
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Changes in 5′-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5′-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5′-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5′-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5′-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5′-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.
4
Biswas, Nabanita, Marta Rodriguez-Garcia, Zheng Shen, Sarah G. Crist, Jack E. Bodwell, John V. Fahey, and Charles R. Wira. "Effects of Tenofovir on Cytokines and Nucleotidases in HIV-1 Target Cells and the Mucosal Tissue Environment in the Female Reproductive Tract." Antimicrobial Agents and Chemotherapy 58, no. 11 (August 18, 2014): 6444–53. http://dx.doi.org/10.1128/aac.03270-14.
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ABSTRACTTenofovir (TFV) is a reverse transcriptase inhibitor used in microbicide preexposure prophylaxis trials to prevent HIV infection. Recognizing that changes in cytokine/chemokine secretion and nucleotidase biological activity can influence female reproductive tract (FRT) immune protection against HIV infection, we tested the hypothesis that TFV regulates immune protection in the FRT. Epithelial cells, fibroblasts, CD4+T cells, and CD14+cells were isolated from the endometrium (Em), endocervix (Cx), and ectocervix (Ecx) following hysterectomy. The levels of proinflammatory cytokines (macrophage inflammatory protein 3α [MIP-3α], interleukin 8 [IL-8], and tumor necrosis factor alpha [TNF-α]), the expression levels of specific nucleotidases, and nucleotidase biological activities were analyzed in the presence or absence of TFV. TFV influenced mRNA and/or protein cytokines and nucleotidases in a cell- and site-specific manner. TFV significantly enhanced IL-8 and TNF-α secretion by epithelial cells from the Em and Ecx but not from the Cx. In contrast, in response to TFV, IL-8 secretion was significantly decreased in Em and Cx fibroblasts but increased with fibroblasts from the Ecx. When incubated with CD4+T cells from the FRT, TFV increased IL-8 (Em and Ecx) and TNF-α (Cx and Ecx) secretion levels. Moreover, when incubated with Em CD14+cells, TFV significantly increased MIP-3α, IL-8, and TNF-α secretion levels relative to those of the controls. In contrast, nucleotidase biological activities were significantly decreased by TFV in epithelial (Cx) and CD4+T cells (Em) but increased in fibroblasts (Em). Our findings indicate that TFV modulates proinflammatory cytokines, nucleotidase gene expression, and nucleotidase biological activity in epithelial cells, fibroblasts, CD4+T cells, and CD14+cells at distinct sites within the FRT.
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Skladanowski, A. C., G. B. Sala, and A. C. Newby. "Inhibition of IMP-specific cytosolic 5′-nucleotidase and adenosine formation in rat polymorphonuclear leucocytes by 5′-deoxy-5′-isobutylthio derivatives of adenosine and inosine." Biochemical Journal 262, no. 1 (August 15, 1989): 203–8. http://dx.doi.org/10.1042/bj2620203.
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1. The partially purified IMP-specific cytosolic 5′-nucleotidases from rat liver, polymorphonuclear leucocytes and heart were inhibited by 50% by 2-6 mM-5′-deoxy-5′-isobutylthioadenosine (IBTA) or 7-10 mM-5′-deoxy-5′-isobutylthioinosine (IBTI). IBTA and IBTI inhibited the rat liver and polymorphonuclear-leucocyte enzymes non-competitively. IBTA, but not IBTI, also inhibited the ecto-5′-nucleotidase of polymorphonuclear leucocytes. IBTI was, by contrast, a more potent inhibitor than IBTA of the AMP-specific soluble 5′-nucleotidase from pigeon heart. 2. During 2-deoxyglucose-induced ATP-catabolism in rat polymorphonuclear leucocytes, adenosine formation was inhibited by approx. 80% by 3 mM-IBTA and by approx. 70% by 7 mM-IBTI. 3. The results show that 5′-modified nucleosides are inhibitors of cytosolic 5′-nucleotidases and that they penetrate to inhibit their target enzymes in intact cells. Such inhibitors may be useful to clarify the mechanisms of adenosine formation and to prevent mononucleotide hydrolysis during ATP breakdown.
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Aslam, Nazia, Syeda Fatima, Sofia Khalid, Shahzad Hussain, Mughal Qayum, Khurram Afzal, and Muhammad Hassham Hassan Bin Asad. "Anti-5 ′ -Nucleotidases (5 ′ -ND) and Acetylcholinesterase (AChE) Activities of Medicinal Plants to Combat Echis carinatus Venom-Induced Toxicities." BioMed Research International 2021 (February 4, 2021): 1–10. http://dx.doi.org/10.1155/2021/6631042.
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Echis carinatus is one of the highly venomous snakes of Pakistan that is responsible for numerous cases of envenomation and deaths. In Pakistan, medicinal plants are commonly used traditionally for snakebite treatment because of their low cost and easy availability in comparison with antivenom. The current research is aimed at evaluating the inhibitory activity of Pakistani medicinal plants against acetylcholinesterase and 5 ′ -nucleotidases present in Echis carinatus venom. Acetylcholinesterase and 5 ′ -nucleotidase enzymatic assays were performed at different venom concentrations to check the activity of these enzymes. Methanolic extracts from different parts of plants were used for in vitro determination of their inhibitory activity against 5 ′ -nucleotidases in snake venom. Active methanolic extracts were subsequently fractioned using different solvents, and these fractions were also assessed for their anti-5 ′ -nucleotidase activity. Results of this study exhibited that Eugenia jambolana Willd. ex O. Berg, Rubia cordifolia L., Trichodesma indicum (L.) R. Br., Calotropis procera (Wild.) R. Br., Curcuma longa L., and Fagonia arabica L. were able to significantly ( p > 0.5 ) neutralize the 5 ′ -nucleotidase activity by 88%, 86%, 86%, 85%, 83.7%, and 83%, respectively, compared with a standard antidote (snake venom antiserum). Thus, this study indicates that these plants possess the potential to neutralize one of the toxic enzymatic components of Echis carinatus venom and hence can help to augment the future efforts of developing alternative therapy for the management of snakebites.
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Skladanowski, A. C., R. T. Smolenski, M. Tavenier, J. W. de Jong, M. H. Yacoub, and A. M. Seymour. "Soluble forms of 5'-nucleotidase in rat and human heart." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 4 (April 1, 1996): H1493—H1500. http://dx.doi.org/10.1152/ajpheart.1996.270.4.h1493.
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Intracellular AMP hydrolysis probably produces sufficient adenosine in ischemic heart to exert physiological activity. Because data on adenosine-producing systems in human heart are scarce, we measured 1) formation of adenosine (catabolites) in ischemic human heart slices and 2) cytoplasmic 5'-nucleotidase activity in human left ventricle. We also measured the latter in rat ventricle and cardiomyocytes. During the first 5 min of incubation, adenosine production in slices (n = 5) equaled 26 +/- 10 (SD) nmol.min-1.g wet wt-1, and total AMP content was 0.81 +/- 0.46 mM. Cytoplasmic IMP-preferring 5'-nucleotidase activity in homogenates of human heart (N-II, 167 +/- 78 mU/g, n = 23) was significantly higher than that of the AMP-preferring one (N-I, 107 +/- 61 mU/g, n = 24). Both isozymes were two to three times more active in rat heart than in human heart. Rat cardiomyocytes contained comparable amounts of the two 5'-nucleotidases. Kinetics of N-I isolated from explanted human heart displayed features similar to the enzyme from animal heart, with a Michaelis constant of 1.5 mM under maximally stimulated conditions. This form can provide the amount of adenosine found in ischemic slices. In conclusion, human heart shows lower cytosolic 5'-nucleotidase activities than rat heart. Nevertheless, cytosolic 5'-nucleotidase activity in human heart can easily account for adenosine formation during ischemia.
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Borowiec, Agnieszka, Katarzyna Lechward, Kinga Tkacz-Stachowska, and Andrzej C. Składanowski. "Adenosine as a metabolic regulator of tissue function: production of adenosine by cytoplasmic 5'-nucleotidases." Acta Biochimica Polonica 53, no. 2 (June 12, 2006): 269–78. http://dx.doi.org/10.18388/abp.2006_3339.
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Adenosine is a product of complete dephosphorylation of adenine nucleotides which takes place in various compartments of the cell. This nucleoside is a significant signal molecule engaged in regulation of physiology and modulation of the function of numerous cell types (i.e. neurons, platelets, neutrophils, mast cells and smooth muscle cells in bronchi and vasculature, myocytes etc.). As part a of purinergic signaling system, adenosine mediates neurotransmission, conduction, secretion, vasodilation, proliferation and cell death. Most of the effects of adenosine help to protect cells and tissues during stress conditions such as ischemia or anoxia. Adenosine receptors and nucleoside transporters are targets for potential drugs in many pathophysiological situations. The adenosine-producing system in vertebrates involves a cascade dephosphorylating ATP and ending with 5'-nucleotidase (EC 3.1.3.5) localized either on the membrane or inside the cell. In this paper the cytoplasmic variants of 5'-nucleotidase are broadly characterized as well as their clinical relevance. The role of AMP-selective 5'-nucleotidase (cN-I) in the heart, skeletal muscle and brain is highlighted. cN-I action is crucial during ischemia and important for the efficacy of some nucleoside-based drugs and in the regulation of the substrate pool for nucleic acids synthesis. Inhibitors used in studying the roles of cytoplasmic and membrane-bound 5'-nucleotidases are also described.
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Piec, G., and M. Le Hir. "The soluble ‘low-Km’ 5′-nucleotidase of rat kidney represents solubilized ecto-5′-nucleotidase." Biochemical Journal 273, no. 2 (January 15, 1991): 409–13. http://dx.doi.org/10.1042/bj2730409.
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A soluble ‘low-Km’ 5′-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5′-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble ‘low-Km‘ 5′-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5′-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble ‘low-Km’ 5′-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5′-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5′-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5′-nucleotidase. The soluble ‘low-Km’ 5′-nucleotidase, like the ecto-5′-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble ‘low-Km’ 5′-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5′-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.
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Moses, G. C., J. F. Tuckerman, and A. R. Henderson. "Biological variance of cholinesterase and 5'-nucleotidase in serum of healthy persons." Clinical Chemistry 32, no. 1 (January 1, 1986): 175–77. http://dx.doi.org/10.1093/clinchem/32.1.175.
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Abstract We measured cholinesterase (EC 3.1.1.8) and 5'-nucleotidase (EC 3.1.3.5) activities in serum of 24 healthy laboratory staff during 12 months. Overall mean activities ranged from 5.3 to 13.4 kU/L for cholinesterase and 5.4 to 9.8 U/L for 5'-nucleotidase. Cholinesterase activity was significantly (p less than 0.01) higher for men than for women. 5'-Nucleotidase activity was significantly (p = 0.01) higher for subjects 40 years or older than for those younger than 40, but was not different with respect to sex or time of year. Average intra- and interindividual variances (SD2) were 0.38 and 2.69 for cholinesterase and 1.41 and 0.97 for 5'-nucleotidase, respectively. Intra- to interindividual standard deviation ratios were 0.38 for cholinesterase and 1.21 for 5'-nucleotidase. Average within-run analytical variances were 0.13 and 0.3 (4% and 13% of total variance) for cholinesterase and 5'-nucleotidase, respectively. The importance of these findings in regards to diagnostic interpretation of serum cholinesterase and 5'-nucleotidase results is discussed.
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Chiarelli, Laurent R., Paola Bianchi, Elisa Fermo, Alessandro Galizzi, Paolo Iadarola, Andrea Mattevi, Alberto Zanella, and Giovanna Valentini. "Functional analysis of pyrimidine 5′-nucleotidase mutants causing nonspherocytic hemolytic anemia." Blood 105, no. 8 (April 15, 2005): 3340–45. http://dx.doi.org/10.1182/blood-2004-10-3895.
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Abstract Inherited pyrimidine 5′-nucleotidase type I (P5′N-1) deficiency is the third most common erythrocyte enzymopathy that causes hemolysis. Fourteen different mutations have been identified to date. We have investigated the molecular bases of the disease by studying the biochemical properties of the recombinant wild-type human enzyme and 4 variant proteins (D87V, L131P, N179S, and G230R) bearing missense mutations found in patients affected by nonspherocytic hemolytic anemia. P5′N-1 is a relatively stable protein and has essentially identical catalytic efficiency toward cytidine monophosphate (CMP) and uridine monophosphate (UMP). All investigated mutant proteins display impaired catalytic properties and/or reduced thermostability, providing a rationale for the pathological effects of the mutations. Despite the substantial changes in the kinetic and thermostability parameters, the enzyme activity detected in the red blood cells of patients homozygous for mutations L131P and G230R exhibits moderate alterations. This suggests that P5′N-1 deficiency is compensated, possibly by other nucleotidases or alternative pathways in nucleotide metabolism. Therefore, nucleotidase activity may not be considered a prognostic indicator in patients affected by the enzymopathy. (Blood. 2005;105:3340-3345)
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Olsson, Ray A. "CardiovascularEcto-5′-Nucleotidase." Circulation Research 95, no. 8 (October 15, 2004): 752–53. http://dx.doi.org/10.1161/01.res.0000146278.94064.4b.
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Panteghini, M. "Electrophoretic fractionation of 5'-nucleotidase." Clinical Chemistry 40, no. 2 (February 1, 1994): 190–96. http://dx.doi.org/10.1093/clinchem/40.2.190.
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Abstract Human isonucleotidases were separated by electrophoresis on a cellulose acetate membrane. Three 5'-nucleotidase forms, NTP1, NTP2, and NTP3, were resolved with this method and quantified by densitometry. The procedure was not only simple and rapid but also sufficiently precise (between-run CV < 20%) and sensitive (detected nucleotidase fractions of > 0.5 U/L). The effects of various treatments (heat, neuraminidase, glycosidases, proteases, lectins, and detergents) on the electrophoretic pattern of 5'-nucleotidase were studied. NTP1 (mean 12% of total 5'-nucleotidase, SD 5%), NTP2 (mean 30%, SD 8%), and NTP3 (mean 58%, SD 8%) were found in all normal persons studied. The increase in total 5'-nucleotidase in patients with hepatobiliary disease was mainly due to the NTP1 isoform.
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Harb, J., K. Meflah, and S. Bernard. "Structural differences between plasma-membrane 5′-nucleotidase in different cell types as evidenced by antibodies." Biochemical Journal 232, no. 3 (December 15, 1985): 859–62. http://dx.doi.org/10.1042/bj2320859.
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Antibodies raised against bovine 5′-nucleotidase inhibit this enzyme as well as 5′-nucleotidase from other bovine tissues, showing common structure(s) between these proteins. However, an IgG fraction directed against the glucidic moiety of the liver enzyme did not cross-react with the enzyme from lymphocyte or caudate nuclei, a clear indication that within the same species the 5′-nucleotidase differs from one cell type to another. In addition, immunoblots after electrophoresis show that the previous antibodies recognize 5′-nucleotidase from human, mouse or chicken origin. However, only human 5′-nucleotidase activity can be inhibited by the antibodies. Thus at least three groups of antigenic determinants must exist on the 5′-nucleotidase: one related to the glucidic moiety of the glycoprotein whose binding inhibits the enzyme activity, another related to the catalytic site, as its binding also led to enzyme inhibition, and a last one of structural nature. It seems that the third group of determinant is common to many species, whereas the second one is more restricted.
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Bengis-Garber, Carmela. "Membrane-bound 5′-nucleotidase in marine luminous bacteria: biochemical and immunological properties." Canadian Journal of Microbiology 31, no. 6 (June 1, 1985): 543–48. http://dx.doi.org/10.1139/m85-101.
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A novel 5′-nucleotidase previously described in halophilic Vibrio costicola was detected in marine Vibrio and Photobacterium strains. The enzyme of marine bacteria was similar in its properties to the 5′-nucleotidase of Vibrio costicola; it was outwardly oriented in the cytoplasmic membrane and dephosphorylated nucleoside 5′-tri-, di-, and mono-phosphates to respective nucleosides before uptake. The enzyme in marine strains was immunologically cross-reactive with the antibody raised against the purified 5′-nucleotidase of Vibrio costicola. The uptake of the products of ATP hydrolysis was studied in Vibrio harveyi, and it was shown that both adenosine and inorganic phosphate released upon the action of 5′-nucleotidase were rapidly taken up by the cell.
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Darvish, A., and P. J. Metting. "Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart." American Journal of Physiology-Heart and Circulatory Physiology 264, no. 5 (May 1, 1993): H1528—H1534. http://dx.doi.org/10.1152/ajpheart.1993.264.5.h1528.
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The major enzyme responsible for adenosine production during myocardial hypoxia or ischemia is 5'-nucleotidase. We purified an AMP-specific 5'-nucleotidase to homogeneity from the 150,000-g supernatant of dog heart homogenate using phosphocellulose, DEAE-cellulose, and ADP-agarose affinity chromatography. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified enzyme yielded a single protein band of 43 kDa. The molecular mass of the holoenzyme, determined by gel filtration and sucrose density-gradient centrifugation, was approximately 166 kDa, suggesting a tetrameric structure. Dog heart cytosolic 5'-nucleotidase was active at physiological pH (6.8-7.8) and demonstrated a preference for AMP over IMP as substrate. The enzyme exhibited sigmoidal saturation kinetics, with half-maximal activity at 2.6 mM AMP in the absence of ADP. ADP (0-250 microM) activated cytosolic 5'-nucleotidase by increasing maximal velocity and affinity for AMP. The enzyme was inhibited by 4 mM ATP, but 5'-nucleotidase activity increased as [ATP] was reduced. Mg2+ was required for activity, with maximal activation at approximately 3.5 mM free Mg2+. These data suggest that the regulation of AMP-specific cytosolic 5'-nucleotidase by adenine nucleotides and free Mg2+ may be important in the production of adenosine during conditions promoting ATP hydrolysis, such as myocardial hypoxia or ischemia.
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Minamino, T., M. Kitakaze, T. Morioka, K. Node, K. Komamura, H. Takeda, M. Inoue, M. Hori, and T. Kamada. "Cardioprotection due to preconditioning correlates with increased ecto-5'-nucleotidase activity." American Journal of Physiology-Heart and Circulatory Physiology 270, no. 1 (January 1, 1996): H238—H244. http://dx.doi.org/10.1152/ajpheart.1996.270.1.h238.
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We investigated whether loss of myocardial protection after ischemic preconditioning (IP) is related to the extent of deactivation of activated ecto-5'-nucleotidase. The coronary arteries of mongrel dogs were occluded four times for 5 min separated by 5 min of reperfusion (IP). Five (IP1), 30 (IP2), 60 (IP3), and 120 min (IP4) after the fourth 5-min coronary occlusion or after a corresponding nonischemic period (control groups), the coronary arteries were occluded for 90 min followed by 6 h of reperfusion. The infarct size-limited effect of IP gradually disappeared in the IP2 (21.6 +/- 3.9%) and IP3 (33.8 +/- 3.6%) groups compared with the IP1 group (8.3 +/- 1.6%) and returned to the control level in the IP4 group (39.9 +/- 5.2%). The increased ecto-5' -nucleotidase activity due to the IP procedure decreased according to the order of IP1 to IP4 groups. Infarct size was inversely correlated with ecto-5'-nucleotidase activity (P < 0.001). An inhibitor of ecto-5'-nucleotidase blunted the infarct size-limiting effect of IP. The infarct size-limiting effect of IP decreased as the activation of ecto-5'-nucleotidase was blunted. These results suggest that ecto-5'-nucleotidase activity plays a key role in the cardioprotection of IP.
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Loe, D. W., J. R. Glover, S. Head, and F. J. Sharom. "Solubilization, characterization, and detergent interactions of lymphocyte 5′-nucleotidase." Biochemistry and Cell Biology 67, no. 4-5 (April 1, 1989): 214–23. http://dx.doi.org/10.1139/o89-033.
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5′-Nucleotidase is a member of a recently identified class of membrane proteins that is anchored via a phosphatidylinositol-containing glycolipid. The enzyme was readily solubilized with full retention of catalytic activity by nonionic and anionic detergents such as alkylthioglucosides, deoxycholate, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS), while the cationic detergent dodecyltrimethylammonium bromide (DTAB) caused loss of activity. 5′-Nucleotidase was released only at high detergent concentrations, suggesting that it is tightly associated with the membrane. DTAB and deoxycholate caused a loss of heat stability, while alkylthioglucosides had no effect. CHAPS produced a remarkable increase in the heat stability of the partially purified (glycoprotein fraction) and purified enzyme. Arrhenius plots of solubilized 5′-nucleotidase showed "break points" for all detergents in the temperature range 30–37 °C. SDS-PAGE of pure 5′-nucleotidase showed a single subunit of molecular mass 70 kilodaltons (kDa), while sucrose density gradient sedimentation gave a peak of activity corresponding to 132 kDa, indicating that the enzyme exists as a dimer. Gel filtration of the solubilized enzyme in several detergents showed apparent molecular masses between 200–630 kDa, suggesting that lymphocyte 5′-nucleotidase may be present in high molecular mass aggregates in its native state.Key words: 5′-nucleotidase, plasma membrane, detergents, solubilization, stability, activation energy.
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Le Hir, M., and U. C. Dubach. "An ATP-inhibited soluble 5'-nucleotidase of rat kidney." American Journal of Physiology-Renal Physiology 254, no. 2 (February 1, 1988): F191—F195. http://dx.doi.org/10.1152/ajprenal.1988.254.2.f191.
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Hydrolysis of 5'-AMP by 5'-nucleotidase is a possible source of adenosine in the kidney. A renal membrane-bound ecto-5'-nucleotidase has been previously described. The present study deals with the catalytic properties of a 5'-AMP phosphohydrolase partially purified from high-speed supernatants of rat kidney homogenates. It exhibits phosphatase activity toward 5'-AMP, 5'-IMP, and 5'-GMP, but not toward 2'- and 3'-AMP and corresponds therefore to a 5'-nucleotidase. The hydrolysis of 5'-AMP by the soluble 5'-nucleotidase requires divalent cations. Maximal activity is reached with 10 microM of either Mn2+ or Co2+, whereas half-maximal activity is obtained with approximately 400 microM Mg2+. The soluble 5'-nucleotidase exhibits Michaelis-Menten kinetics with a Km of 9.5 microM for 5'-AMP. In the presence of 1 mM of free Mg2+, physiological concentrations of ATP provoke an increase of the Km for 5'-AMP and a decrease of Vmax. An increase of the pH of 0.4 units in the pH range 6.4-7.4 roughly doubles the rate of hydrolysis of 5'-AMP. The effects of ATP and of the pH are compatible with a role of the renal soluble 5'-nucleotidase in the hydrolysis of 5'-AMP and in the production of adenosine during hypoxia.
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Opdebeeck, Britt, Isabel R. Orriss, Ellen Neven, Patrick C. D’Haese, and Anja Verhulst. "Extracellular Nucleotides Regulate Arterial Calcification by Activating Both Independent and Dependent Purinergic Receptor Signaling Pathways." International Journal of Molecular Sciences 21, no. 20 (October 15, 2020): 7636. http://dx.doi.org/10.3390/ijms21207636.
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Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5′-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor—pyrophosphate and the calcification stimulator—inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization.
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Garcia-Gil, Mercedes, Marcella Camici, Simone Allegrini, Rossana Pesi, Edoardo Petrotto, and Maria Tozzi. "Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors." International Journal of Molecular Sciences 19, no. 11 (November 14, 2018): 3598. http://dx.doi.org/10.3390/ijms19113598.
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The growing evidence of the involvement of purine compounds in signaling, of nucleotide imbalance in tumorigenesis, the discovery of purinosome and its regulation, cast new light on purine metabolism, indicating that well known biochemical pathways may still surprise. Adenosine deaminase is important not only to preserve functionality of immune system but also to ensure a correct development and function of central nervous system, probably because its activity regulates the extracellular concentration of adenosine and therefore its function in brain. A lot of work has been done on extracellular 5′-nucleotidase and its involvement in the purinergic signaling, but also intracellular nucleotidases, which regulate the purine nucleotide homeostasis, play unexpected roles, not only in tumorigenesis but also in brain function. Hypoxanthine guanine phosphoribosyl transferase (HPRT) appears to have a role in the purinosome formation and, therefore, in the regulation of purine synthesis rate during cell cycle with implications in brain development and tumors. The final product of purine catabolism, uric acid, also plays a recently highlighted novel role. In this review, we discuss the molecular mechanisms underlying the pathological manifestations of purine dysmetabolisms, focusing on the newly described/hypothesized roles of cytosolic 5′-nucleotidase II, adenosine kinase, adenosine deaminase, HPRT, and xanthine oxidase.
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Le Hir, M. "A soluble 5′-nucleotidase in rat kidney. Stimulation by decavanadate." Biochemical Journal 273, no. 3 (February 1, 1991): 795–98. http://dx.doi.org/10.1042/bj2730795.
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A soluble 5′-nucleotidase was identified in rat kidney and partially purified. Compared with 5′-IMP, 5′-AMP was a poor substrate. The affinity for 5′-IMP was very low (S0.5 greater than 1 mM) in the absence of an activator, and it was much increased (S0.5 = 0.1 mM) by 2,3-bisphosphoglycerate (2,3-DPG). ATP and bisadenosyl tetraphosphate were further activators. The pH optimum was 6.3. Those properties suggest that the renal soluble 5′-nucleotidase is identical with the ‘high-Km’ 5′-nucleotidase purified previously from liver, heart and erythrocytes. Decavanadate (100 nM) increased the rate of hydrolysis of 1 mM-5′-IMP 16-fold. The effect was specific for the decameric form of vanadate, since it was not reproduced by either decavanadate-free orthovanadate or pervanadate. Half-maximal activation was obtained at 1.4 nM-decavanadate. Decavanadate increased the affinity of the soluble 5′-nucleotidase for 5′-IMP. The effects of 2,3-DPG and of vanadate were not additive. Thus decavanadate probably influences the soluble 5′-nucleotidase in the same way as 2,3-DPG, but with a much higher potency.
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Rees, David C., John A. Duley, and Anthony M. Marinaki. "Pyrimidine 5′ Nucleotidase Deficiency." British Journal of Haematology 120, no. 3 (February 2003): 375–83. http://dx.doi.org/10.1046/j.1365-2141.2003.03980.x.
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Rampazzo, Chiara, Cinzia Gazziola, Paola Ferraro, Lisa Gallinaro, Magnus Johansson, Peter Reichard, and Vera Bianchi. "Human high-Km5′-nucleotidase." European Journal of Biochemistry 261, no. 3 (May 1999): 689–97. http://dx.doi.org/10.1046/j.1432-1327.1999.00320.x.
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Itoh, Roichi. "IMP-GMP 5′-nucleotidase." Comparative Biochemistry and Physiology Part B: Comparative Biochemistry 105, no. 1 (May 1993): 13–19. http://dx.doi.org/10.1016/0305-0491(93)90163-y.
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Pesi, R., M. Turriani, S. Allegrini, C. Scolozzi, M. Camici, P. L. Ipata, and M. G. Tozzi. "The Bifunctional Cytosolic 5′-Nucleotidase: Regulation of the Phosphotransferase and Nucleotidase Activities." Archives of Biochemistry and Biophysics 312, no. 1 (July 1994): 75–80. http://dx.doi.org/10.1006/abbi.1994.1282.
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LEHTO, Marty T., and Frances J. SHAROM. "Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5′-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer." Biochemical Journal 332, no. 1 (May 15, 1998): 101–9. http://dx.doi.org/10.1042/bj3320101.
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Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5´-nucleotidase (5´-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5´-nucleotidase all obeyed Michaelis–Menten kinetics, with a Km for 5´-AMP in the range 11–16 µM. The GPI anchor was removed from essentially all 5´-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5´-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5´-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5´-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5´-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.
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Baron, M. D., B. Pope, and J. P. Luzio. "The membrane topography of ecto-5′-nucleotidase in rat hepatocytes." Biochemical Journal 236, no. 2 (June 1, 1986): 495–502. http://dx.doi.org/10.1042/bj2360495.
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The transmembrane topography of the rat hepatocyte ectoenzyme 5′-nucleotidase was studied by the use of glycoprotein labelling and limited-proteolysis techniques. Comparison, by one-dimensional peptide mapping, of enzyme iodinated from outside the cell with that iodinated in the solubilized state showed that no additional iodination sites were revealed on solubilization. Incubation of newly synthesized enzyme in a microsomal membrane fraction with proteinase showed that the entire molecule of 5′-nucleotidase was protected from proteolysis. These data suggest that little, if any, of the 5′-nucleotidase molecule is present on the cytoplasmic side of the plasma membrane. No evidence was found for a previously proposed interaction between 5′-nucleotidase and actin, although the ability of preparations of 5′-nucleotidase to prevent inhibition of deoxyribonuclease I by actin was explained by minute traces of ATPase activity. Comparison of peptide maps of enzyme labelled by iodination or by methods specific for carbohydrate showed that in both cases predominantly one section of the molecule was labelled. It is proposed that the enzyme is a short-stalked integral membrane protein without a cytoplasmic domain in which about one-third of the molecule forms the accessible molecular surface.
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Stochaj, U., K. Flocke, W. Mathes, and H. G. Mannherz. "5′-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol–glycan." Biochemical Journal 262, no. 1 (August 15, 1989): 33–40. http://dx.doi.org/10.1042/bj2620033.
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We have analysed the membrane anchorage of plasma-membrane 5′-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5′-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5′-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5′-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5′-nucleotidase were detected as both soluble and membrane-bound forms.
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Sharom, Frances J., Ian Lorimer, and Mary P. Lamb. "Reconstitution of lymphocyte 5′-nucleotidase in lipid bilayers: behaviour and interaction with concanavalin A." Canadian Journal of Biochemistry and Cell Biology 63, no. 10 (October 1, 1985): 1049–57. http://dx.doi.org/10.1139/o85-130.
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Pure 5′-nucleotidase (EC 3.1.3.5) and a membrane glycoprotein fraction (partially purified 5′-nucleotidase) were isolated from pig lymphocyte plasma membrane by affinity chromatography techniques, using the cationic detergent dodecyltrimethylammonium bromide as a solubilizing agent. A detergent-dialysis technique was used to reconstitute both partially purified and pure enzyme into large unilamellar phospholipid vesicles, where it remains functional. 5′-Nucleotidase is relatively unstable in detergent solutions, but is highly stable once reconstituted into lipid vesicles. Arrhenius plots of the enzyme in bilayers of dimyristoyl phosphatidylcholine show a break point at 22–23 °C, with a different activation energy above and below the phospholipid gel-to-liquid crystalline phase transition. 5′-Nucleotidase in intact plasma membrane is inhibited more than 95% by concanavalin A in a positively cooperative fashion (Hill coefficient = 2.1), as is partially purified reconstituted enzyme. Purification of the enzyme before reconstitution results in less than 50% inhibition by concanavalin A and a complete loss of positive cooperativity (Hill coefficient < 1.0). The inhibition properties of the enzyme can be fully restored by co-reconstituting pure 5′-nucleotidase with the remaining lymphocyte glycoproteins.
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Sharom, Frances J., Mary P. Lamb, Christine C. Kupsh, and Susan Head. "Inhibition of lymphocyte 5′-nucleotidase by lectins: effects of lectin specificityand cross-linking ability." Biochemistry and Cell Biology 66, no. 7 (July 1, 1988): 715–23. http://dx.doi.org/10.1139/o88-082.
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5′-Nucleotidase, an integral glycoprotein enzyme of the lymphocyte plasma membrane, is inhibited cooperatively by the lectin concanavalin A. Because divalent succinyl-concanavalin A is a poor enzyme inhibitor, both binding and lectin-induced cross-linking of 5′-nucleotidase may be necessary for inhibition. Succinyl-concanavalin A does not compete with concanavalin A for binding to the enzyme; however, maleyl-concanavalin A, another poor inhibitor, competes effectively with the parent lectin. Thus, maleyl-concanavalin A binds to the same site as concanavalin A but causes little inhibition, whereas succinyl-concanavalin A does not bind to this site. The monovalent lectin from Ricinus communis (RCA-60) is a more effective enzyme inhibitor than the related divalent lectin (RCA-120), and inactivation of the second low-affinity sugar binding site on RCA-60 does not abolish inhibition, suggesting that multivalent cross-linking is not required for 5′-nucleotidase inhibition. Peanut and wheat germ agglutinins do not inhibit the enzyme, whereas lectins from lentil, pea, soybean, Griffonia simplicifolia, and Phaseolus vulgaris inhibit 5′-nucleotidase with various degrees of effectiveness. The only lectin showing strong positive cooperativity in its interaction with 5′-nucleotidase is concanavalin A.
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Vogel, M., H. J. Kowalewski, H. Zimmermann, A. Janetzko, R. U. Margolis, and H. E. Wollny. "Association of the HNK-1 epitope with 5′-nucleotidase from Torpedo marmorata (electric ray) electric organ." Biochemical Journal 278, no. 1 (August 15, 1991): 199–202. http://dx.doi.org/10.1042/bj2780199.
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5′-Nucleotidase isolated from the electric organ of the electric ray (Torpedo marmorata) has a molecular mass of 62 kDa and, on two-dimensional electrophoresis, separates into up to 13 isoforms within a pI range of 5.9-6.7. The N-terminal sequence data show a 71% identity over 17 amino acids with that previously published for the rat liver enzyme. All forms of 5′-nucleotidase are recognized by the HNK-1 monoclonal antibody. HNK-1 immunoreactivity is found at the surface of the Schwann-cell processes covering the synaptic terminals and in this respect corresponds to that of 5′-nucleotidase in the same tissue. Since a number of glycoproteins involved in cell recognition and cell adhesion carry the HNK-1 epitope, 5′-nucleotidase may play a role in cell-cell or cell-extracellular matrix interaction in addition to its activity as an enzyme.
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ENNES-VIDAL, V., R. O. S. CASTRO, C. BRITTO, H. BARRABIN, C. M. D'AVILA-LEVY, and O. C. MOREIRA. "CrATP interferes in the promastigote-macrophage interaction in Leishmania amazonensis infection." Parasitology 138, no. 8 (June 17, 2011): 960–68. http://dx.doi.org/10.1017/s0031182011000710.
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SUMMARYRecent have shown the relationship between Ecto-Nucleoside-Triphosphate-Diphosphohydrolases (Ecto-NTPDases or ecto-nucleotidases) and virulence and infectivity in trypanosomatids. In this work, the inhibition of the ecto-ATPase activities and promastigote growth of Leishmania amazonensis by CrATP was characterized. Furthermore, this compound was used to investigate the role of ecto-nucleotidase in the interaction of L. amazonensis with resident peritoneal macrophages obtained from BALB/c mice. CrATP partially inhibits the ecto-ATPase activity, presenting Ki values of 575·7±199·1 and 383·5±79·0 μm, in the presence or absence of 5 mm MgCl2, respectively. The apparent Kms for ATP (2·9±0·5 mm to Mg2+-dependent ecto-ATPase and 0·4±0·2 mm to Mg2+-independent ecto-ATPase activities) are not significantly altered by CrATP, suggesting a reversible non-competitive inhibition of both enzymes. When CrATP was added to the cultivation medium at 500 μm, it drastically inhibited the cellular growth. The interaction of promastigote forms of L. amazonensis with BALB/c peritoneal macrophages is strongly affected by CrATP. When the parasites were treated with 500 μm CrATP before interacting with macrophages, the adhesion and endocytic indices were strongly reduced to 53·0±14·8% and 39·8±1·1%, respectively. These results indicate that ecto-nucleotidase plays an important role in the infection process caused by Leishmania amazonensis.
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McMILLEN, Lyle, Ifor R. BEACHAM, and Dennis M. BURNS. "Cobalt activation of Escherichia coli 5'-nucleotidase is due to zinc ion displacement at only one of two metal-ion-binding sites." Biochemical Journal 372, no. 2 (June 1, 2003): 625–30. http://dx.doi.org/10.1042/bj20021800.
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Escherichia coli 5′-nucleotidase activity is stimulated 30- to 50-fold in vitro by the addition of Co2+. Seven residues from conserved sequence motifs implicated in the catalytic and metal-ion-binding sites of E. coli 5′-nucleotidase (Asp41, His43, Asp84, His117, Glu118, His217 and His252) were selected for modification using site-directed mutagenesis of the cloned ushA gene. On the basis of comparative studies between the resultant mutant proteins and the wild-type enzyme, a model is proposed for E. coli 5′-nucleotidase in which a Co2+ ion may displace the Zn2+ ion at only one of two metal-ion-binding sites; the other metal-ion-binding site retains the Zn2+ ion already present. The studies reported herein suggest that displacement occurs at the metal-ion-binding site consisting of residues Asp84, Asn116, His217 and His252, leading to the observed increase in 5′-nucleotidase activity.
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Cabezas, Alicia, Iralis López-Villamizar, María Jesús Costas, José Carlos Cameselle, and João Meireles Ribeiro. "Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB." Molecules 26, no. 8 (April 16, 2021): 2307. http://dx.doi.org/10.3390/molecules26082307.
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The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.
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Lilleker, J. B., A. Rietveld, S. R. Pye, K. Mariampillai, O. Benveniste, M. T. J. Peeters, J. A. L. Miller, et al. "Cytosolic 5′-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis." Annals of the Rheumatic Diseases 76, no. 5 (January 25, 2017): 862–68. http://dx.doi.org/10.1136/annrheumdis-2016-210282.
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ObjectivesAutoantibodies directed against cytosolic 5′-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5′-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis.Materials and methodsData from various European inclusion body myositis registries were pooled. Anticytosolic 5′-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression.ResultsData from 311 patients were available for analysis; 102 (33%) had anticytosolic 5′-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients.InterpretationDifferences were observed in clinical and histopathological features between anticytosolic 5′-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5′-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.
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Newby, A. C. "The pigeon heart 5′-nucleotidase responsible for ischaemia-induced adenosine formation." Biochemical Journal 253, no. 1 (July 1, 1988): 123–30. http://dx.doi.org/10.1042/bj2530123.
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1. A 5′-nucleotidase with a strong preference for AMP over IMP was characterized in homogenates and subcellular fractions of pigeon heart by using concentrations of ATP, ADP and AMP which mimicked those present in the ischaemic tissue. 2. The AMP-5′-nucleotidase had a neutral pH optimum and an apparent Km in the range 4.6-5.2 mM. It was stimulated by ATP plus ADP, and was inhibited by other nucleoside monophosphates, Pi and p-nitrophenyl phosphate, but not by ribose 5-phosphate or beta-glycerophosphate. The enzyme was not inhibited by [alpha beta-methylene] ADP or by 5′-deoxy-5′-isobutylthioadenosine, an inhibitor of the previously purified IMP-preferring cytosolic 5′-nucleotidase. 3. Subcellular-fractionation studies indicated that the enzyme has access to cytosolic AMP, although it may be associated by weak ionic interactions with an organelle present in the low-speed particulate fraction. 4. A 5′-nucleotidase was detected under similar conditions in homogenates of rat heart. 5. The activity of the pigeon heart AMP-5′-nucleotidase was sufficient to account for previously measured rates of ischaemia-induced adenosine formation. The similar activity in rat heart could, however, account for only part of ischaemia-induced adenosine formation in this tissue.
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Sala-Newby, Graciela B., Nicola V. E. Freeman, Maria A. Curto, and Andrew C. Newby. "Metabolic and functional consequences of cytosolic 5′-nucleotidase-IA overexpression in neonatal rat cardiomyocytes." American Journal of Physiology-Heart and Circulatory Physiology 285, no. 3 (September 2003): H991—H998. http://dx.doi.org/10.1152/ajpheart.00053.2003.
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Adenosine exerts a spectrum of energy-preserving actions on the heart negative chronotropic effects. The pathways leading to adenosine formation have remained controversial. In particular, although cytosolic 5′-nucleotidases can catalyze adenosine formation in cardiomyocytes, their contribution to the actions of adenosine has not been documented previously. We recently cloned two closely related AMP-preferring cytosolic 5′-nucleotidases (cN-IA and -IB); the A form predominates in the heart. In this study, we overexpressed pigeon cN-IA in neonatal rat cardiomyocytes using an adenovirus. cN-IA overexpression increased adenosine formation and release into the medium caused by simulated hypoxia and by isoproterenol in the absence and presence of inhibitors of adenosine metabolism. Adenosine release was not affected by an ecto-5′-nucleotidase inhibitor, α,β-methylene-ADP, but was affected by a nucleoside transporter, dipyridamole. The positive chronotropic effect of isoproterenol (130 ±3 vs. 100 ±4 beats/min) was inhibited (107 ±3 vs. 94 ±3 beats/min) in cells overexpressing cN-IA, and this was reversed by the addition of the adenosine receptor antagonist 8-( p-sulfophenyl)theophilline (120 ± 3 vs. 90 ± 4 beats/min). Our results demonstrate that overexpressed cN-IA can be sufficiently active in cardiomyocytes to generate physiologically effective concentrations of adenosine at its receptors.
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Bailyes, E. M., M. A. J. Ferguson, C. A. Colaco, and J. P. Luzio. "Inositol is a constituent of detergent-solubilized immunoaffinity-purified rat liver 5′-nucleotidase." Biochemical Journal 265, no. 3 (February 1, 1990): 907–9. http://dx.doi.org/10.1042/bj2650907.
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myo-Inositol analysis of detergent-solubilized immunoaffinity-purified rat liver 5′-nucleotidase showed the presence of 1 mol of myo-inositol/mol of enzyme monomer. This provides unequivocal evidence that the ectoenzyme 5′-nucleotidase is attached to liver membranes by a glycosyl-phosphatidylinositol lipid anchor.
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Spychala, J., V. Madrid-Marina, P. J. Nowak, and I. H. Fox. "AMP and IMP dephosphorylation by soluble high- and low-Km 5'-nucleotidases." American Journal of Physiology-Endocrinology and Metabolism 256, no. 3 (March 1, 1989): E386—E391. http://dx.doi.org/10.1152/ajpendo.1989.256.3.e386.
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Three distinct 5'-phosphomonoesterase activities were isolated from soluble fractions of human placenta, cultured human T and B lymphoblasts, and rat liver using 5'-AMP-sepharose 4B affinity chromatography. We define these activities as "low-Km" 5'-nucleotidase, "high-Km" 5'-nucleotidase, and nonspecific phosphatase. High-Km 5'-nucleotidase was eluted with 0.5 M NaCl, low-Km 5'-nucleotidase was eluted with 10 mM ADP, and nonspecific phosphatase was not retained on the column. We have found significant variability in the relative content of high- to low-Km activities in the tissues studied with the ratios ranging from 5.5 to 264. The properties were studied after further purification. The molecular mass of the low-Km enzymes ranged from 72.5 to 209 kDa, optimum pH ranged from 7.4 to 9.0, Km for AMP ranged from 7 to 15 microM, and Km for IMP ranged from 10 to 26 microM. The molecular mass of the high-Km enzymes ranged from 182 to 210 kDa, pH optimum was at 6.5, Km for AMP ranged from 3.0 to 9.4 mM, and the Km for IMP ranged from 0.3 to 0.5 mM. The data indicate that the soluble low- and high-Km 5'-nucleotidase coexist in the mammalian cells and tissues studied. These observations suggest a complex system for the regulation of nucleoside 5'-monophosphate dephosphorylation.
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ALLEGRINI, Simone, Rossana PESI, Maria Grazia TOZZI, J. Carol FIOL, B. Robert JOHNSON, and Staffan ERIKSSON. "Bovine cytosolic IMP/GMP-specific 5′-nucleotidase: cloning and expression of active enzyme in Escherichia coli." Biochemical Journal 328, no. 2 (December 1, 1997): 483–87. http://dx.doi.org/10.1042/bj3280483.
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A cDNA coding for bovine cytosolic IMP/GMP-specific 5ʹ-nucleotidase endowed with phosphotransferase activity was cloned from calf thymus RNA, by 5ʹ and 3ʹ rapid amplification of cDNA ends protocols (5ʹ and 3ʹ RACE). Two products were isolated: a 5ʹ RACE 1.6 kb fragment and a 3ʹ RACE 2.0 kb fragment, with an overlapping region of 505 bp, leading to a total length of approx. 2951 bp. The similarity in the coding region to that of the human 5ʹ-nucleotidase cDNA sequence [Oka, Matsumoto, Hosokawa and Inoue (1994) Biochem. Biophys. Res. Commun. 205, 917-922], indirectly identified as a 5ʹ-nucleotidase, was 94% and the deduced amino acid sequences were 99.5% identical. The bovine cDNA sequence included the sequences codifying for six peptides obtained from 5ʹ-nucleotidase/phosphotransferase purified from calf thymus. Northern blots of human mRNA species from different tissues showed a 3.6 kb mRNA expressed at equal levels in most tissues. The cDNA was cloned into a pET-28c expression vector and the protein obtained after induction had a molecular mass of 61 kDa under SDS/PAGE. It exhibited both 5ʹ-nucleotidase and phosphotransferase activity, as well as immunological and kinetic properties similar to those of the enzyme purified from calf thymus. This is the first time that a fully active recombinant 5ʹnucleotidase has been described.
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Walker, Jon P., John C. Barbato, and Lauren Gerard Koch. "Cardiac adenosine production in rat genetic models of low and high exercise capacity." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 283, no. 1 (July 1, 2002): R168—R173. http://dx.doi.org/10.1152/ajpregu.00621.2001.
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We previously demonstrated that Copenhagen (COP) and DA inbred rat strains show a wide difference in a test for aerobic treadmill running that correlated positively with isolated cardiac function. The purpose of this study was to test adenosine production as a candidate intermediate phenotype that may explain part of the difference in running and cardiac performance in these genetic models for low and high aerobic capacity. Adenosine production was measured as the activity of soluble 5′-nucleotidase and membrane-bound ecto-5′-nucleotidase in the membrane pellet and supernatant fractions of left and right ventricular muscle and gracilis muscle taken from 10 DA and 10 COP rats. Ecto-5′-nucleotidase activity in the membrane pellet of hearts from both DA and COP accounted for the vast majority of the total tissue adenosine production (>90% in the left ventricle and >80% in the right ventricle). Ecto-5′-nucleotidase activity in the pellet fraction was significantly higher in the left (22.4%) and right (46.1%) ventricles of DA rats compared with COP rats, with no differences in total protein content. There were no significant differences between the strains for 5′-nucleotidase activity in the cardiac supernatant, the gracilis pellet, or the gracilis supernatant. These data support the hypothesis that an increase in cardiac adenosine production may contribute to the greater aerobic running capacity of the DA rats.
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Bontemps, F., G. Van den Berghe, and H. G. Hers. "5′-Nucleotidase activities in human erythrocytes. Identification of a purine 5′-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate." Biochemical Journal 250, no. 3 (March 15, 1988): 687–96. http://dx.doi.org/10.1042/bj2500687.
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A purine 5′-nucleotidase has been separated by DEAE-Trisacryl chromatography from other 5′-nucleotidase activities present in human haemolysates and purified approx. 30,000-fold by subsequent chromatography on Blue Sepharose. The enzyme has an Mr of around 250,000, displays hyperbolic substrate-saturation kinetics and hydrolyses preferentially IMP, GMP and their deoxy counterparts. It is much less active with AMP and dAMP. The purine 5′-nucleotidase is inhibited by Pi, and is strongly stimulated by ATP, dATP and GTP, and by glycerate 2,3-bisphosphate. Stimulators decrease Km and increase Vmax. Glycerate 2,3-bisphosphate is the most potent stimulator of the enzyme and, under physiological conditions, over-rides the influence of the other effectors. Glycerate 2,3-bisphosphate also influences the binding of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood. It is concluded that the glycerate 2,3-bisphosphate-stimulated purine 5′-nucleotidase is responsible for the dephosphorylation of IMP and GMP, but not of AMP, in human erythrocytes.
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Faudry, Eric, Silene P. Lozzi, Jaime M. Santana, Marian D'Souza-Ault, Sylvie Kieffer, Carlos R. Felix, Carlos A. O. Ricart, Marcelo V. Sousa, Thierry Vernet, and Antonio R. L. Teixeira. "Triatoma infestansApyrases Belong to the 5′-Nucleotidase Family." Journal of Biological Chemistry 279, no. 19 (February 25, 2004): 19607–13. http://dx.doi.org/10.1074/jbc.m401681200.
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Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bugTriatoma infestanswas achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes fiveN-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5′-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase fromAedes aegyptiand with 5′-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5′-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity inT. infestanscould represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.
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Malin, Edyth L., and Jay J. Basch. "Differential release of proteins from bovine fat globule membrane." Biochemistry and Cell Biology 68, no. 5 (May 1, 1990): 899–902. http://dx.doi.org/10.1139/o90-133.
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Alkaline phosphatase and 5′-nucleotidase are covalently linked to phosphatidylinositol in bovine fat globule membrane, as demonstrated by their release following treatment with phospholipase C specific for phosphatidylinositol. The failure of this treatment to liberate phosphodiesterase I may indicate that it has a variant linkage resistant to release. In a test of exposure at the membrane surface, alkaline phosphatase and phosphodiesterase I, but not 5′-nucleotidase, were released from fat globule membrane by treatment with proteinase K. These apparent differences in accessibilities of membrane surface proteins suggest that attachment to phosphatidylinositol does not necessarily impart greater exposure to proteins with which it is linked.Key words: alkaline phosphatase, 5′-nucleotidase, phosphodiesterase I, phosphatidylinositol, phospholipase C, fat globule membrane.
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Roszek, Katarzyna, Katarzyna Bomastek, Marian Drożdżal, and Michał Komoszyński. "Dramatic differences in activity of purines metabolizing ecto-enzymes between mesenchymal stem cells isolated from human umbilical cord blood and umbilical cord tissue." Biochemistry and Cell Biology 91, no. 6 (December 2013): 519–25. http://dx.doi.org/10.1139/bcb-2013-0050.
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The high quality human mesenchymal stem cells (MSCs) with remarkable expansion potential in culture are demonstrated to possess multifold clinical applications. However, their isolation and characterization are difficult and sometimes ambiguous. We exploited nucleotide metabolizing ecto-enzymes for more complete characterization of MSCs. Using standard methods of cell culturing and analyses, we detected significant differences in the activity of ecto-nucleotidases on the surface of MSCs isolated from umbilical cord tissue and MSC-like cells derived from umbilical cord blood. Interestingly, the proliferation rate and the immunophenotypic characteristics of mesenchymal stem cells also correspond to the activities of these enzymes. Compared with the CD90-, CD105-, and CD73-deficient and slowly proliferating UCB-MSC-like cells that had relatively higher ecto-NTPDases activity, the CD90-, CD105-, and CD73-positive and rapidly proliferating UC-MSCs rather had ecto-5′-nucleotidase activity and presented neither ecto-nucleotidases nor adenylate kinase activities. In summary, our results demonstrate for the first time that activity of purine nucleotide metabolizing ecto-enzymes differs significantly between mesenchymal stem cells drawn from different neonatal sources, corresponding with a distinct proliferative potential.
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DOLESKI, PEDRO H., RICARDO E. MENDES, DANIELA B. R. LEAL, NATHIELI B. BOTTARI, MANOELA M. PIVA, ESTER S. DA SILVA, MATEUS E. GABRIEL, et al. "Seric and hepatic NTPDase and 5′ nucleotidase activities of rats experimentally infected byFasciola hepatica." Parasitology 143, no. 5 (March 1, 2016): 551–56. http://dx.doi.org/10.1017/s0031182015001882.
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SUMMARYThe enzymatic activities of NTPDase and 5′nucleotidase are important to regulate the concentration of adenine nucleotides, known molecules involved in many physiological functions. Therefore, the objective of this study was to evaluate the activity of NTPDase and 5′nucleotidase in serum and liver tissue of rats infected byFasciola hepatica. Rats were divided into two groups: uninfected control and infected. NTPDase activity for adenosine triphosphate (ATP) and ADP substrates in the liver was higher compared with the control group at 15 days post-infection (PI), while seric activity was lower. In addition, seric and hepatic samples did not show changes for 5′nucleotidase activity at this time. On the other hand, either NTPDase or 5′nucleotidase activities in liver homogenate and serum were higher at 87 days PI. Early in the infection, low NTPDase activity maintains an increase of ATP in the bloodstream in order to activate host immune response, while in hepatic tissue it decreases extracellular ATP to maintain a low inflammatory response in the tissue. As stated, higher NTPDase and 5′nucleotidase activities 87 days after infection in serum and tissue, probably results on an increased concentration of adenosine molecule which stimulates a Th2 immune response. Thus, it is possible to conclude thatF. hepaticainfections lead to different levels of nucleotide degradation when considering the two stages of infection studied, which influences the inflammatory and pathological processes developed by the purinergic system.
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Dai, Qin-xue, Shan Li, Miao Ren, Xinlu Wu, Xin-yu Yao, Fei-hong Lin, Xu-qing Ni, Yun-chang Mo, and Jun-lu Wang. "Analgesia with 5' extracellular nucleotidase-mediated electroacupuncture for neuropathic pain." Arquivos de Neuro-Psiquiatria 80, no. 3 (March 2022): 289–95. http://dx.doi.org/10.1590/0004-282x-anp-2021-0149.
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ABSTRACT Background: Acupuncture is a treatment for neuropathic pain, but its mechanism remains unclear. Previous studies showed that analgesia was induced in rats with neuropathic pain when their spinal cord adenosine content increased after electroacupuncture (EA); however, the mechanism behind this electroacupuncture-induced increase has not been clarified. Objective: This study aimed to determine the role that ecto-5’-nucleotidase plays in EA-induced analgesia for neuropathic pain. Methods: We performed electroacupuncture at the Zusanli acupoint on the seventh day after establishing a rat model of neuropathic pain induced through chronic constriction injuries. We observed the mechanical withdrawal threshold and thermal pain threshold and detected the expression of ecto-5’-nucleotidase in the spinal cord using Western blot. Chronic constriction injury rat models were intraperitoneally injected with α,β-methyleneadenosine 5'-diphosphate, an ecto-5’-nucleotidase inhibitor, 30 min before electroacupuncture. The adenosine content of the spinal cord was detected using high-performance liquid chromatography. Lastly, the adenosine A1 receptor agonist N6-cyclopentyladenosine was intrathecally injected into the lumbar swelling of the rats, and the mechanical withdrawal and thermal pain thresholds were reevaluated. Results: Analgesia and increased ecto-5’-nucleotidase expression and adenosine content in the spinal cord were observed 1 h after electroacupuncture. α,β-methyleneadenosine 5'-diphosphate was able to inhibit upregulation of adenosine content and electroacupuncture-induced analgesia. After administration of N6-cyclopentyladenosine, electroacupuncture-induced analgesia was restored. Conclusions: Our results suggest that electroacupuncture at Zusanli can produce analgesia in chronic constriction injury rat models, possibly via the increased ecto-5’-nucleotidase expression induced through electroacupuncture, thus leading to increased adenosine expression in the spinal cord.
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Willadsen, P., J. M. Nielsen, and G. A. Riding. "Purification and properties of a novel nucleotide-hydrolysing enzyme (5′-nucleotidase) from Boophilus microplus." Biochemical Journal 258, no. 1 (February 15, 1989): 79–85. http://dx.doi.org/10.1042/bj2580079.
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The tick Boophilus microplus contains a nucleoside phosphate-hydrolysing enzyme which, in many respects, resembles the well characterized 5'-nucleotidase from mammalian tissue. The tick enzyme has been purified to homogeneity. It is a membrane-bound glycoprotein with an apparent Mr of 67,000 and, although it fails to hydrolyse a range of nucleoside 2'- or 3'-monophosphates, it has broad specificity for the 5' derivatives. Further investigation of the enzyme's substrate specificity, however, shows some important differences from the mammalian nucleotidases. It hydrolyses both bis-p-nitrophenyl phosphate and p-nitrophenyl phenylphosphonate, typical substrates for phosphodiesterases. However, the tick enzyme is most strikingly different from the mammalian enzymes in that it hydrolyses not only AMP but ADP and ATP as well. Further, the products of the hydrolysis of ATP are adenosine and tripolyphosphate, a reaction which has not been reported previously. The products of ADP hydrolysis are adenosine and pyrophosphate.
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Özsoylu, Şinasi. "About Pyrimidine 5’-Nucleotidase Deficiency." Turkish Journal of Hematology 30, no. 2 (June 5, 2013): 227. http://dx.doi.org/10.4274/tjh.2013.0050.
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