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PubMed1983

Ecto-5'-nucleotidase does not catalyze vectorial production of adenosine in the perfused rat liver.

From the results obtained in perfused rat hearts, Frick and Lowenstein proposed that ecto-5'-nucleotidase catalyzes a vectorial reaction in which AMP hydrolysis is accompanied by transfer of adenosine across the cell membrane (Frick, G.P., and Lowenstein, J.M. (1978) J. Biol. Chem. 253, 1240-1244). We have examined by the use of perfused rat livers, the uptake mechanism of adenosine generated from AMP by ecto-5'-nucleotidase. Recirculating perfusion of rat livers was performed with a buffered saline containing [2-3H]AMP at an initial concentration of 5 microM. One-half of the [2-3H] AMP was dephosphorylated by 0.8 min of perfusion; less than 13% of the radioactivity of hydrolyzed [2-3H] AMP was located in [2-3H]adenosine plus [2-3H]inosine appearing in the perfusate. Addition of 6-[(4-nitrobenzyl)thio]-guanosine, an inhibitor of the nucleoside transport system, at 120 microM to the perfusate caused a 3.9-fold increase in the amount of the [2-3H]AMP-derived 3H-nucleosides appearing in the perfusate. Moreover, in a perfusion in which uridine was added to the perfusate at 2.6 mM to compete with the [2-3H] AMP-derived [2-3H]adenosine for the nucleoside transport system, more than 87% of the radioactivity of hydrolyzed [2-3H]AMP was located in [2-3H]adenosine appearing in the perfusate; the result indicates that the uridine added to the perfusate efficiently trapped the [2-3H]adenosine formed from [2-3H]AMP. These results support an uptake mechanism of the AMP-derived adenosine in which the adenosine formed by ecto-5'-nucleotidase in the blood-sinusoidal plasma membrane of hepatocytes, is taken up by the nucleoside transport system located at the plasma membrane side by side with ecto-5'-nucleotidase. The results therefore indicate that the ecto-5'-nucleotidase of hepatocytes does not catalyze vectorial production of adenosine, in contrast to the previous report on perfused rat hearts.

5'-Nucleotidase↗

PubMed1984

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro.

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.0 nmol/hr/10(6) cells, which is the same as the activity in cord blood lymphocytes (7.0 +/- 2 nmol/hr/10(6) cells). The activity rose twofold to 13.2 +/- 3.8 nmol/hr/10(6) cells at 6 mo of age (p less than 0.001, paired t-test), which is similar to the activity in adult peripheral blood lymphocytes (14.1 +/- 6.3 nmol/hr/10(6) cells). This increased activity in total lymphocytes reflects increased activity in the B cell population. B cell ecto-5'-nucleotidase activity in two infants at 12 to 13 mo of age was 19.3 and 25.2 nmol/hr/10(6) cells, values that are four-to fivefold higher than for cord blood B cells (5.6 +/- 2.8 nmol/hr/10(6) cells) and within the normal range for adult B cells (27.9 +/- 12 nmol/hr/10(6) cells). In spite of a greatly expanded peripheral blood B cell population, studies of immunoglobulin biosynthesis in vitro demonstrated that infant peripheral blood B cells are functionally immature with no synthesis of IgG in response to Epstein Barr virus. Thus, the increase in peripheral blood B lymphocyte ecto-5'-nucleotidase activity in infants precedes their acquisition of a capacity for IgG synthesis in vitro. Data from a hypogammaglobulinemic infant revealed a persistently low ecto-5'-nucleotidase activity over a 10-mo period until at 14 mo of age the activity was 8.8 nmol/hr/10(6) cells in total lymphocytes and 13.0 nmol/hr/10(6) cells in B cells, which correlated with in vivo and in vitro evidence of delayed B cell maturation. Thus, ecto-5'-nucleotidase activity may be a useful cell surface marker in studies of human postnatal B cell maturation.

5'-Nucleotidase↗

PubMed1984

Absence of lymphocyte ecto-5'-nucleotidase in infants with reticuloendotheliosis and eosinophilia (Omenn's syndrome).

Lymphocytes from three infants with reticuloendotheliosis and eosinophilia ( Omenn 's syndrome) and immunodeficiency were assayed for 5'-nucleotidase activity. B and T lymphocytes from all three patients were totally deficient in ecto-5'-nucleotidase activity, but had normal levels of cytoplasmic 5'-nucleotidase. In contrast, cultured lymphocytes expressed normal ectoplasmic and cytoplasmic activities, suggesting that the lymphocyte-restricted enzyme deficiency was not likely a primary genetic defect. The deficiency of lymphocyte ecto-5'-nucleotidase was not associated with any abnormality of deoxynucleoside metabolism. The absence of lymphocyte ecto-5'-nucleotidase may be a characteristic feature of this syndrome and may help to distinguish this disease from others with similar manifestations.

5'-Nucleotidase↗

PubMed2005

Modulation of ecto-5'-nucleotidase by phospholipids in human umbilical vein endothelial cells (HUVEC).

Ecto-5'-nucleotidase, the major enzyme controlling extracellular adenosine production, can be activated by phospholipids, e.g. lysophosphatidylcholine (LPC). This study examined the structural requirements of phospholipids to evoke this enzyme activation and figured out two new activators of ecto-5'-nucleotidase: platelet activating factor (PAF) and sphingosylphosphorylcholine (SPC). Potential signal transduction pathways including an involvement of protein kinase C and PAF-receptor were evaluated on the model of human umbilical vein endothelial cells (HUVEC). Cells were pre-incubated with 10 microM of various phospholipids including lysophosphatidylcholine, beta-arachidonyl-gamma-palmityl-alpha-phosphatidylcholine, beta,gamma-dipalmityl-alpha-phosphatidyl-choline, beta,gamma-dipalmityl-alpha-phosphatidylethanolamine, beta,gamma-dipalmityl-alpha-phosphatidylserine, gamma-acyl-beta-lyso-alpha-phosphatidylethanolamine, beta-acetyl-gamma-O-hexadecyl-alpha-phosphatidylcholine (platelet activating factor), lysophosphatidylic acid, sphingosine-1-phosphate and sphingosylphosphorylcholine. In the cell supernatant the extracellular dephosphorylation rate of the fluorescent AMP-analogue 1,N6-etheno-5'AMP to 1,N6-etheno-adenosine was measured by HPLC. Out of these ten structurally related phospholipids only lysophosphatidylcholine, sphingosylphosphatidylcholine and platelet activating factor dose-dependently increased the activity of ecto-5'-nucleotidase. Pharmacological blocking experiments revealed that neither the activation of PAF-receptor nor of protein kinase C were important for mediating the activation of ecto-5'-nucleotidase. Thus, using information on the known molecular structures of tested phospholipids, a phosphatidylcholine residue in alpha-position and a short chain length fatty acid esterified in beta-position seem essential for activation of ecto-5'-nucleotidase by glycerophospholipids. Since all tested phospholipids have similar fatty acid chain lengths and residues in alpha-position, they should act similarly on membrane fluidity. It is concluded that the observed effects are not based on changes in membrane fluidity by the added phospholipids, but rather involve a yet to be determined phospholipid-receptor.

5'-Nucleotidase↗

PubMed1985

Nucleotidase activities of human peripheral lymphocytes.

Several B lymphoblastic cell lines are known to be relatively resistant to the combination of 2'-deoxyadenosine with an adenosine deaminase inhibitor. These cell lines are believed to have a greater capacity to dephosphorylate 2'-deoxyadenosine nucleotides, thus preventing excessive accumulation of potentially toxic metabolites. In this study, the 2'-deoxynucleoside 5'-monophosphate dephosphorylating activities of human peripheral lymphocytes were examined. Peripheral lymphocytes have at least three nucleotide 5'-monophosphate nucleotidases distinguished by different pH optimums, substrate preference, Mg2+ requirement, inhibitors, and molecular weights. Two of the enzymes appeared to be cytosolic, only one of which had significant substrate activity with dAMP. This enzyme had an acidic pH optimum (5.0), no Mg2+ requirement, was inhibited by tartrate, and demonstrated broad substrate specificity. The other cytosolic nucleotidase required Mg2+, had a pH optimum of 5.5 to 6.0, was activated by 2'-deoxyinosine, and demonstrated a substrate preference for 3'- and 5'-monophosphate 2'-deoxynucleosides of hypoxanthine, guanine, uracil, and thymine. The third enzyme, ecto 5'-nucleotidase, is associated with the cell membrane. Although the ecto 5'-nucleotidase activity was higher in the B lymphocytes, the cytosolic nucleotidases were similar in activity in the T and B lymphocytes.

B-Lymphocytes↗

PubMed1993

Adenosine stimulates 5'-nucleotidase activity in rat mesangial cells via A2 receptors.

Because A2 adenosine receptor activation stimulates adenylate cyclase and cyclic AMP induces 5'-nucleotidase expression in rat mesangial cells, we examined the effect of adenosine and its analogs on 5'-nucleotidase activity in these cells. A2 adenosine receptors were characterized using [3H]5'-N-ethylcarboxamidoadenosine (NECA) as a tracer. There was a single group of receptor sites with a KD value of 0.53 microM and a number of sites of 1,317 fmol/mg. [3H]NECA binding was inhibited preferentially by A2 adenosine analogs and antagonists. Similarly, the order of potency for cAMP stimulation was in favour of A2 adenosine analogs. Rat mesangial cells expressed surface 5'-nucleotidase activity. Exposure of cells for 48 h to adenosine analogs showed that at low concentrations A2 analogs stimulated 5'-nucleotidase activity. These results indicate that adenosine upregulates activity of 5'-nucleotidase, the enzyme responsible for its local formation, via A2 receptor stimulation and increase in cAMP production.

5'-Nucleotidase↗

PubMed1988

Cytosolic purine 5'-nucleotidases of rat liver and human red blood cells: regulatory properties and role in AMP dephosphorylation.

Of the various species of cellular 5'-nucleotidases, membranous, lysosomal and cytosolic, only the latter are likely to play a role in the physiologic dephosphorylation of the 5'-nucleoside monophosphates present in the cytoplasm. The necessity to preserve cellular ATP renders a strict control of the dephosphorylation as well as of the deamination of AMP mandatory, because both nucleotides are maintained in equilibrium by adenylate kinase. Our studies of cytosolic purine 5'-nucleotidases purified from rat liver and from human erythrocytes, reviewed in this presentation, have shown that both display complex kinetic properties. Both enzymes have markedly higher affinities for IMP and for GMP than for AMP. In addition, they are stimulated by nucleoside triphosphates, among them ATP and GTP, and inhibited by Pi. The erythrocytic purine 5'-nucleotidase is also stimulated by glycerate 2,3-bisphosphate. It could thus be expected that under conditions of ATP and GTP breakdown, particularly when accompanied by an increase in Pi, the dephosphorylation of AMP would be curtailed. To verify this hypothesis, experiments were performed with isolated rat hepatocytes and with human red blood cells. The rate of dephosphorylation of AMP was measured by following time-wise the production of adenosine in the presence of coformycin (or deoxycoformycin) and 5-iodotubercidin. The coformycins inhibit the deamination of adenosine into inosine by adenosine deaminase, and 5-iodotubercidin inhibits the recycling of adenosine into AMP by adenosine kinase. Upon induction of ATP catabolism by the addition of fructose to isolated rat hepatocytes, the dephosphorylation of AMP was nearly completely suppressed. In accordance with these results, the activity of the rat liver cytosolic 5'-nucleotidase, assayed in the presence of concentrations of substrate and effectors mimicking those measured in intact cells following the addition of fructose, was decreased as compared to control conditions. In hepatocytes in which ATP catabolism was induced by suppression of oxygen, the rate of dephosphorylation of AMP increased about 3-fold. However, in contradiction with these data, the activity of the cytosolic 5'-nucleotidase, measured under conditions mimicking anoxia, decreased markedly. In human erythrocytes, dephosphorylation of AMP did not occur under physiologic conditions, but proceeded when ATP catabolism was induced by glucose lack or by alkalinization. The rate of dephosphorylation of AMP was 3-fold higher during glucose deprivation than under alkaline conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Monophosphate↗

PubMed1982

Evidence for a continual exchange of 5'-nucleotidase between the cell surface and cytoplasmic membranes in cultured rat fibroblasts.

Approximately 40% of the 5'-nucleotidase activity in cultured rat embryo fibroblasts was patent, as judged by enzymatic assays comparing the activity of intact cells with detergent-solubilized cells. The patent activity was inhibited when cells were incubated with anti-5'-nucleotidase serum at 2 degrees C, whereas latent activity (calculated as the difference between total and patent activity) was not. Latent activity was inhibited by antibody when the antiserum was added directly to detergent-solubilized cells or when cells were cultured in the presence of antiserum for several hours. Patent activity was inhibited by antibody, and cells were returned to culture in antibody-free medium; after 12 hr, 30% of the total activity was expressed in intact cells and 60% of the anti-5'-nucleotidase, assayed by the binding of sheep antirabbit antibodies to intact cells, was lost from the cell surface, indicating an exchange of 5'-nucleotidase between the latent and patent compartments. Cytochemical studies showed that the patent activity was located on the cell surface and that latent activity was present in cytoplasmic vacuoles and vesicles, and in the Golgi complex. Over 30% of the anti-5'-nucleotidase internalized during 6 hr in culture returned to the cell surface after a further 9 hr, indicating a continual exchange of the enzyme between the cell surface and cytoplasmic membranes.

Animals↗

PubMed1995

A re-evaluation of the ultrastructural localization of 5'-nucleotidase activity in the developing rat cerebellum, with a cerium-based method.

The membrane ectoenzyme 5'-nucleotidase converts 5'-AMP into adenosine which, in the nervous tissue, plays an important role as intercellular messenger. Moreover, during histogenesis, 5'-nucleotidase seems to be related to cell proliferation and migration. Conflicting data are reported in the literature about the localization (neuronal or glial) of 5'-nucleotides in the rat cerebellum. In the present report we have analyzed the distribution of 5'-nucleotidase activity with electron microscopy, using a cerium-based method, at different postnatal histogenetic stages (postnatal days (PND) 11, 17, 28). On PND 11 and 17, rims of reaction product outlined the plasma membranes of some neuroblasts in the external granular layer and of parallel fibers and some migrating cells in the developing molecular layer. Positivity was frequently observed on membranes of adjacent neuronal cells and glial processes. Moderate activity was also present on the membranes of granule cells and of mossy fiber rosettes and granule cell dendrites constituting the cerebellar glomeruli within the internal granule cell layer. At PND 28, the reaction product was slightly reduced in some localizations. Cytochemical patterns prove that the cerium-based method is suitable for demonstration of 5'-nucleotidase-specific activity. In fact, a continuous and fine reaction product appears strictly linked to the cell membranes, and no unevenly scattered precipitates can be observed. Data suggest that, during cerebellar histogenesis, 5'-nucleotidase may be involved in the mechanisms of cell migration and proliferation. However, in adulthood, prominent localization of the reaction product on neuronal elements suggests a major role in neuromodulation processes for the enzyme.

5'-Nucleotidase↗

PubMed1991

Deoxyadenosine-resistant human T lymphoblasts with elevated 5'-nucleotidase activity.

Although several different enzymes with 5'-nucleotidase activity have been described in mammalian cells, their functions in nucleotide metabolism have not been clearly distinguished. In the present experiments, a mutant human T lymphoblastoid cell line (CEM-dAdoR) was selected specifically for resistance to deoxyadenosine toxicity. Compared to parental CEM cells, the variant had 4-fold elevated ATP-activated cytosolic 5'-nucleotidase activity. Other enzymes of potential importance for deoxyadenosine metabolism were indistinguishable in the two cell types. In medium supplemented with the adenosine deaminase inhibitor deoxycoformycin, the T cells with increased 5'-nucleotidase accumulated less nucleotides from exogenously added deoxyadenosine, or 9-beta-D-arabinofuranosyladenine, than did parental T lymphocytes. These metabolic changes were associated with resistance to the growth inhibitory effects of these nucleosides, and also to deoxyguanosine and to 9-beta-D-arabinofuranosylguanine. The T cells with elevated 5'-nucleotidase activity formed more 2',3'-dideoxyadenosine than did parental cells, in deoxycoformycin-supplemented medium. The accumulation of 2',3'-dideoxyadenosine 5'-triphosphate from 2',3'-dideoxyinosine was similarly augmented in the mutant. These data establish the importance of the cytosolic 5'-nucleotidase for the metabolism of purine 2'-deoxyribonucleosides, arabinonucleosides and 2',3'-dideoxyribonucleosides in T lymphoblasts.

5'-Nucleotidase↗

PubMed1986

3'-nucleotidase activity in procyclic and bloodstream stages of Trypanosoma rhodesiense.

Both procyclic and bloodstream-derived trypanosomes of Trypanosoma rhodesiense exhibit a nucleotidase activity which is capable of hydrolyzing 3'-ribonucleotides. Nucleotidase activity in assays employing 5'-nucleotide substrates is not detectable in preparations of either phenotype. The 3'-nucleotidase activity in lysates of procyclic trypanosomes is pelletable at 100,000 g, whereas that activity from bloodstream forms is operationally soluble under the conditions employed. The 3'-nucleotidase activities of both stages share the following characteristics: pH optimum of 8.5, substrate preference for purine ribonucleotides, resistance to commonly used phosphatase inhibitors, including fluoride, tartrate and molybdate ions. Both activities are reversibly inhibited by EDTA and reactivated by Co2+ ions. As visualized by fine-structure cytochemical methods, 3'-nucleotidase activity was distributed over the entire surface of procyclic trypanosomes, but not bloodstream parasites bearing an intact surface coat.

Animals↗

PubMed2004

A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis.

We have isolated and expressed a cDNA from the parasitic nematode Trichinella spiralis encoding a novel secreted nucleotidase which catalyses the hydrolysis of nucleoside 5'-diphosphates and 5'-monophosphates, but not 5'-triphosphates. The full length cDNA encodes a protein of 550 amino acids with an N-terminal signal peptide, but lacking a C-terminal signature sequence for addition of a glycosyl phosphatidylinositol (GPI) anchor. Expression in Pichia pastoris resulted in the secretion of an active enzyme with the catalytic properties of both a Mg2+-dependent diphosphohydrolase/apyrase and a 5'-nucleotidase. The protein sequence is homologous to 5'-nucleotidases from a wide variety of organisms but contains no sequences specifically conserved in apyrases, suggesting that it is a representative of a new class of secreted nucleotidase. The enzyme was essentially monospecific for AMP among the nucleoside 5'-monophosphates and catalysed the hydrolysis of nucleoside 5'-diphosphates in the order of UDP >> ADP. The diphosphatase activity was dependent on the presence of magnesium ions and a reducing agent, while the 5'-nucleotidase activity was enhanced by these additions. Kinetic analyses indicated that the enzyme exhibits allosteric behaviour. Determination of the number of active sites suggested that catalysis of the two different reactions occurs at the same active site. The data are discussed in terms of regulation of host purinergic signalling during infection.

5'-Nucleotidase↗

PubMed2006

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

This study describes the enzymatic properties of an ecto-5'-nucleotidase in Trichomonas gallinae. The enzyme hydrolyzes nucleoside monophosphates at pH 7.2 and is activated by divalent cations, such as magnesium. Ecto-5'-nucleotidase activity was insensitive to levamisole, tetramisole (alkaline phosphatase inhibitors), and AMPCP (adenosine 5'-[alpha,beta-methylene]diphosphate), an ecto-5'-nucleotidase inhibitor, whereas 0.1mM ammonium molybdate (considered a potent inhibitor of 5'-nucleotidase activity) completely inhibited the enzyme activity. The apparent K(M) (Michaelis constant) and Vmax (maximum velocity) values for Mg2+-AMP were 466+/-57 microM and 3.7+/-0.59 nmolPi/min/10(6) trichomonads, respectively. Considering that trichomonads lack the ability to synthesize purines and pyrimidines de novo, the presence of an ecto-5'-nucleotidase in intact trophozoites of T. gallinae could be important in regulating the extracellular nucleotide levels and generating adenosine, essential for the survival strategies of the parasite.

5'-Nucleotidase↗

PubMed1977

[5'-Nucleotidase activity of lymphocyte plasma membranes. Effect of concanavalin A].

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.

Adenosine Triphosphatases↗

PubMed1990

Membrane-anchoring domain of rat liver 5'-nucleotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid.

The involvement of glycosylphosphatidylinositol (GPI) in membrane anchoring of 5'-nucleotidase was investigated by chemical analyses. 5'-Nucleotidase purified from rat liver microsomes was subjected to BrCN cleavage, hexane extraction, and high-performance liquid chromatography, resulting in the purification of a single fragment with Mr 2300. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser and characteristic components of GPI including ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. In addition, it was confirmed that digestion of 5'-nucleotidase with lysyl endopeptidase yielded a fragment containing the dipeptide Phe-Ser and the same GPI components as above. The sequences of the tetradeca- and dipeptides thus determined are identified at positions 510-523 and 522-523, respectively, in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing a hydrophobic amino acid sequence [Misumi, Y., Ogata, S., Hirose, S., & Ikehara, Y. (1990) J. Biol. Chem. 265, 2178-2183]. Taken together, these results indicate that the mature 5'-nucleotidase molecule lacks the predicted COOH-terminal peptide extension and is attached at serine-523 with GPI, which functions as the membrane anchor of 5'-nucleotidase.

5'-Nucleotidase↗

PubMed1998

Ecto-5'-nucleotidase from a human colon adenocarcinoma cell line. Correlation between enzyme activity and levels in intact cells.

Differences on 5'-nucleotidase activity in intact Rugli and BCS-TC2 cells (rat glioblastoma and human colon adenocarcinoma cell lines, respectively) are not due to differences in the characteristics of the ectoenzyme. A membrane-bound 5'-nucleotidase from BCS-TC2 cells has been purified to homogeneity with a high specific activity (130 U/mg), yielding a single 72-kDa band on SDS-PAGE. It is a metalloenzyme and, after inhibition by EDTA, its activity can be partially restored by divalent cations. The hydrolysis of the nucleosides 5'-monophosphate used as substrate follows Michaelis-Menten kinetics; ADP and concanavalin A are competitive and non-competitive inhibitors of the AMPase activity, respectively. This ecto-5'-nucleotidase is a high-mannose glycoprotein; deglycosylation converts the 72-kDa into a 59-kDa protein with a concomitant activity loss. The enzyme purified from BCS-TC2 cells shows similar characteristics from that previously isolated from Rugli cells; differences between them are mainly due to glycosylation. Polyclonal antibodies against 5'-nucleotidase from BCS-TC2 cells also show cross-reactivity with the enzyme from Rugli cells. When the ectoenzyme activity is measured in cells in culture, Rugli cells present a higher activity than BCS-TC2 cells however, they express very low amounts of ecto-5'-nucleotidase. Our results also show a reduction in protein level and enzyme activity associated with a decrease in the differentiation degree and an increase in tumorigenicity of human colon adenocarcinoma BCS-TC2 sublines.

5'-Nucleotidase↗

PubMed2000

Effect of shear stress on the release of soluble ecto-enzymes ATPase and 5'-nucleotidase along with endogenous ATP from vascular endothelial cells.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5'-nucleotidases). The activity of soluble ATPase was progressively increased from 1.62+/-0.27 to 12.7+/-1.0 pmoles ml(-1) h(-1) after 60 min of stimulation by shear stress. The rate of [(3)H]-ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid, and remained insensitive to the classic inhibitors of ion-pumping and intracellular ATPases. Shear stress also increased the activity of 5'-nucleotidase in the medium from 2.0+/-0.5 to 27.2+/-2.8 pmoles ml(-1) h(-1). When shear stress was applied after removal of ecto-5'-nucleotidase by phosphatidylinositol-specific phospholipase C, the release of 5'-nucleotidase was drastically reduced. These results show that soluble ATPase and 5'-nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

5'-Nucleotidase↗

PubMed1991

A soluble 5'-nucleotidase in rat kidney. Stimulation by decavanadate.

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.

5'-Nucleotidase↗