Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.
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PMID:Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum. 164 65

The ecto-5'-nucleotidase activity of rat glomerular mesangial cells increases after exposure to prostaglandin E2 (PGE2) via cAMP stimulation (Savic et al., 1990, Immunology 70, 321). Therefore we examined whether other cAMP-stimulating agents had a similar effect. Forskolin (1 microM), PGE2 (10 microM), and isoproterenol (10 microM), three products stimulating rat mesangial cell adenylate cyclase activity, enhanced cAMP accumulation within 5 min and 5'-nucleotidase activity after a lag time of at least 24 h, 3-Isobutyl-1-methylxanthine (IBMX) and Ro 20-1724, two drugs inhibiting cAMP degradation, also stimulated cAMP accumulation and 5'-nucleotidase activity. The effects of these agents on 5'-nucleotidase activity were additive with those of the three products stimulating adenylate cyclase activity, except for Ro 20-1724 and forskolin which acted synergistically. Cycloheximide, a blocker of protein synthesis, suppressed the cAMP-dependent increase of 5'-nucleotidase activity. Because ecto-5'-nucleotidase activity is a marker of cell differentiation, the effect of the same cAMP-stimulating agents on cell proliferation was also studied. Forskolin, PGE2, and isoproterenol inhibited [3H]thymidine incorporation into rat mesangial cells in a dose-dependent manner. The same effect was obtained with IBMX (100 microM) and Ro 20-1724 (50 microM). Stimulation of 5'-nucleotidase activity and inhibition of [3H]thymidine incorporation occurred over the same range of concentrations for the various agonists tested. Taken together, these results indicate that expression of ecto-5'-nucleotidase in rat mesangial cells is induced by cAMP whatever the reason for its accumulation. The simultaneous inhibition of DNA synthesis may occur independently or be associated with the stimulation of 5'-nucleotidase expression.
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PMID:Cyclic adenosine monophosphate-stimulating agents induce ecto-5'-nucleotidase activity and inhibit DNA synthesis in rat cultured mesangial cells. 165 63

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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PMID:The soluble 'low-Km' 5'-nucleotidase of rat kidney represents solubilized ecto-5'-nucleotidase. 184 40

Glycosylation and carbohydrate processing of ecto-5'-nucleotidase were studied in cultured human chorionic cells using metabolic labelling and immunoprecipitation with monoclonal antibodies. Tunicamycin blocks glycosylation altogether leading to a reduction in molecular mass of 9,500 Da. The same result is obtained by digesting the mature 72,000-Da protein with endoglycosidase F. Using various inhibitors of the carbohydrate-trimming reactions like deoxynojirimycin, deoxymannojirimycin and swainsonine smaller molecular mass reductions are observed and the oligosaccharide side chains are kept in a configuration sensitive to endoglycosidase H digestion. Digestion of mature 5'-nucleotidase with endoglycosidase H leads to a much smaller (2,000 Da) reduction in molecular mass. It is calculated that, in addition to the phosphatidylinositol-glycan anchor structure, ecto-5'-nucleotidase of human chorionic cells should carry 4 oligosaccharide side chains per subunit, 3 of which should be of the complex and one of the high mannose type. Interference with carbohydrate processing by various inhibitors does not seem to influence the distribution of ecto-5'-nucleotidase between the cell surface and intracellular membranes nor does it block the transfer of the enzyme to the phosphatidylinositol glycan anchor.
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PMID:Glycosylation and processing of carbohydrate side chains of ecto-5'-nucleotidase in cultured human chorionic cells. 214 Feb 64

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.
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PMID:Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides. 215 57

Because ecto-5'-nucleotidase activity of rat glomerular mesangial cells has been shown to increase upon interaction with macrophages in vitro, it was examined whether interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha), two macrophage-released cytokines, were responsible for this effect. IL-1 beta and TNF-alpha stimulated mesangial cell 5'-nucleotidase activity in a dose-dependent manner after treatment for 24 hr. Maximum increases reached 4.5 times and 1.7 times basal values for IL-1 beta (20 U/ml) and TNF-alpha (25 ng/ml), respectively. The effects of both cytokines were additive. Stimulation of 5'-nucleotidase by IL-1 beta and TNF-alpha was specific since the activity of other ectoenzymes, such as Mg2(+)-ATPase, was unchanged. Cycloheximide, a blocker of protein synthesis, suppressed the cytokine-dependent increase of 5'-nucleotidase activity. Cyclo-oxygenase inhibitors such as indomethacin and ibuprofen inhibited approximately 50% of the effects of both cytokines. Their inhibitory effect was abolished in the presence of prostaglandin E2 (PGE2). In addition, PGE2 itself produced a dose-related (0.1-10 microM) increase of 5'-nucleotidase activity with a maximum of 2.2 times basal value. Taken together, these results indicate that IL-1 beta, essentially, and TNF-alpha, to a lesser extent, regulate 5'-nucleotidase expression in the plasma membrane of cultured mesangial cells and that their effect depends in part on PGE2 synthesis. Therefore, macrophages, via their products of secretion acting on 5'-nucleotidase, could modulate adenosine production in the glomerular capillaries.
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PMID:Induction of ecto-5'-nucleotidase of rat cultured mesangial cells by interleukin-1 beta and tumour necrosis factor-alpha. 216 99

A soluble 5'-nucleotidase was purified 200-fold from pigeon heart. The enzyme (1) had an apparent molecular mass close to 150 kDa, (2) had a neutral pH optimum and hydrolysed a wide range of nucleoside 5'-monophosphates with a 15-fold preference for AMP over IMP, (3) at near-physiological concentrations of AMP was activated by ADP but not by ATP, (4) was inhibited by high Mg2+ concentration and high ionic strength, (5) was weakly inhibited by p-nitrophenol phosphate and Pi, and (6) was non-competitively inhibited more potently by 5'-deoxy-5'-isobutylthioinosine than by 5'-deoxy-5'-isobutylthioadenosine, but not by [alpha,beta-methylene]ADP. The data show that the enzyme is distinct from the ecto-5'-nucleotidase and from the previously purified IMP-specific 5'-nucleotidase. They also predict that the enzyme is activated during ATP catabolism and hence will generate a more-than-linear increase in the adenosine-formation rate in response to an increase in cytosolic AMP concentration.
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PMID:Partial purification and properties of an AMP-specific soluble 5'-nucleotidase from pigeon heart. 234 53

Functional studies were performed on human peripheral blood T lymphocytes stained with goat anti-5'-nucleotidase antibodies and separated into ecto-5'-nucleotidase (ecto-5'-NT)-positive and -negative populations using the FACSTAR fluorescence-activated cell sorter. On the average, ecto-5'-NT+ T cells contained 34 +/- 13% CD4+ and 55 +/- 15% CD8+ cells, whereas ecto-5'-NT-T cells contained 65 +/- 12% CD4+ and 23 +/- 8% CD8+ cells. Staining with anti-5'-NT antibodies did not significantly alter the ability of unseparated T cells to proliferate in response to PHA or PMA, or in a MLR. However, prior incubation with anti-5'-NT antibodies did inhibit the ability of irradiated T cells to provide help for PWM-stimulated Ig synthesis by as much as 55%. In five separate experiments, ecto-5'-NT-T cells demonstrated an equal or better ability to incorporate [3H]TdR after PHA stimulation or in a MLR, as compared with ecto-5'-NT+ T cells. Similarly, ecto-5'-NT- T cells were not diminished in their ability to provide help for autologous B cells in a PWM-driven system. Clearly, the inability of ecto-5'-NT- T cells from patients with a variety of immunodeficiency diseases to function in these assays cannot be explained solely by their lack of ecto-5'-NT activity. In contrast, ecto-5'-NT-positive and -negative T cells showed markedly different dose-response curves for proliferation in response to PMA. Ecto-5'-NT+ T cells responded to lower doses of PMA (1.0 ng/ml) than did ecto-5'-NT- T cells and showed a two- to eight-fold greater rate of [3H]TdR incorporation at 3 to 10 ng of PMA per ml. Ecto-5'-NT+ T cells may have a protein kinase C that is more accessible or more easily activated or may utilize an alternate pathway of activation when stimulated with low concentrations of PMA.
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PMID:Functional characterization of ecto-5'-nucleotidase-positive and -negative human T lymphocytes. 253 56

Human placental ecto-5'-nucleotidase is specifically detected on Western blots using poly- or monoclonal antibodies. In two-dimensional electrophoresis 5'-nucleotidase, purified as well as membrane bound, is resolved in up to 13 isoforms distinguished by a different content of neuraminic acid. These forms span a range of molecular masses of about 67-71 kDa and of isoelectric points of 5.8-7.0. Chemical cross-linking of the purified enzyme with either homoor heterobifunctional reagents yields a dimer of about 140 kDa exclusively. On the other hand treatment of plasma membranes with the same reagents results in a crosslinking product of 5'-nucleotidase of about 97 kDa. The partner of the enzyme subunit in this crosslink, a protein of about 30 kDa, is unknown. Using specific antibodies the cytoskeletal components actin and tropomyosin were excluded as possible candidates.
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PMID:Human placental ecto-5'-nucleotidase: isoforms and chemical crosslinking products of the membrane-bound and isolated enzyme. 254 Jul 66

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.
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PMID:5'-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart. 254 75


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