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PubMed2000

The modulation of ecto-nucleotidase activities by glutamate in cultured cerebellar granule cells.

Several enzymes hydrolyze ATP, producing ADP which is hydrolyzed to AMP. Ecto-5'-nucleotidase produces adenosine from AMP. Glutamate (Glu) is an excitatory neurotransmitter and increases extracellular adenosine levels, which is considered an important inhibitory neuromodulator. Here we show that Glu activates ADP and AMP hydrolysis. NMDA and kainic acid (KA) also increased these enzymatic activities, but 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD) had no effect. Dihydrokainate (DHK), an inhibitor of glutamate uptake, also blocked glutamate-evoked activation of ecto-nucleotidases, suggesting that this activation was also Glu transporters dependent. Therefore, we suggest that the Glu-evoked stimulation of ecto-nucleotidases might contribute to the increase of adenosine in extracellular space induced by Glu.

Adenosine Diphosphate↗
PubMed1983

De novo synthesis of 3'-nucleotidase in germinating wheat embryo.

The enzyme 3'-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up (35)SO(4) between 40 and 46 hours of germination synthesized (35)S-labeled 3'-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3'-nucleotidase activity is a consequence of de novo synthesis of the enzyme.

Journal Article↗
PubMed1988

Nucleoside diphosphatase and 5'-nucleotidase activities of soybean root nodules and other tissues.

Nucleoside diphosphatase and 5'-nucleotidase activities were both found to be very high in extracts of soybean (Glycine max L.) root nodules. Both activities increased early in soybean nodule development, prior to the rise in leghemoglobin, and both were found at equivalent levels in nitrogenfixing and nonfixing nodules. Based on a survey of other tissues, these activities were both highest in soybean nodules (1300 nanomoles per milligram protein per minute, nucleoside diphosphatase and 500 nanomoles per milligram protein per minute, 5'-nucleotidase), but they were not always associated with each other; in some tissues one was high and the other low. Neither activity correlated well with ureide production; both seem, rather, to be primarily involved in some other metabolic function. Both the nucleoside diphosphatase and 5'-nucleotidase of soybean nodules were soluble proteins, and neither appeared to be associated with plastids, mitochondria, or bacteroids.

Journal Article↗
PubMed2003

Rat pancreas secretes particulate ecto-nucleotidase CD39.

In exocrine pancreas, acini release ATP and the excurrent ducts express several types of purinergic P2 receptors. Thereby, ATP, or its hydrolytic products, might play a role as a paracrine regulator between acini and ducts. The aim of the present study was to elucidate whether this acinar-ductal signalling is regulated by nucleotidase(s), and to characterize and localize one of the nucleotidases within the rat pancreas. Using RT-PCR and Western blotting we show that pancreas expresses the full length ecto-nucleoside triphosphate diphosphohydrolase, CD39. Immunofluorescence shows CD39 localization on basolateral membranes of acini and intracellularly. In small intercalated/ interlobular ducts, CD39 immunofluorescence was localized on the luminal membranes, while in larger ducts it was localized on the basolateral membranes. Upon stimulation with cholecystokinin-octapeptide-8 (CCK-8), acinar CD39 relocalizes in clusters towards the lumen and is secreted. As a result, pancreatic juice collected from intact pancreas stimulated with CCK-8 contained nucleotidase activity, including that of CD39, and no detectable amounts of ATP. Anti-CD39 antibodies detected the full length (78 kDa) CD39 in pancreatic juice. This CD39 was confined only to the particulate and not to the soluble fraction of CCK-8-stimulated secretion. No CD39 activity was detected in secretion stimulated by secretin. The role of secreted particulate, possibly microsomal, CD39 would be to regulate intraluminal ATP concentrations within the ductal tree. In conclusion, we show a novel inducible release of full length particulate CD39, and propose its role in the physiological context of pancreatic secretion.

Adenosine Triphosphatases↗
PubMed1991

5'-nucleotidase activity in a eutrophic lake and an oligotrophic lake.

Differences in enzymatic hydrolysis of dissolved organic phosphorus and subsequent phosphorus uptake were compared by using dual-labeled (gamma-P and 2-H) ATP in oligotrophic Lake Michigan and a moderately eutrophic lake in southeastern Michigan. More than 50% of the phosphate that was hydrolyzed was immediately taken up into bacterium-sized particles in the eutrophic lake and at a near-shore site in Lake Michigan. Less than 50% of the hydrolyzed phosphate was taken up into bacterium-sized particles at an offshore site in Lake Michigan. It is hypothesized that differences in size-fractionated uptake were the result of greater phosphorus utilization capacity in bacteria in habitats where loading of organic carbon is greater. Substantial isotope dilution of labeled phosphate uptake by unlabeled phosphate occurred, which implied that the phosphate was hydrolyzed extracellularly in both systems. Comparable nucleotidase activities were measured in the eutrophic lake and Lake Michigan, but the significance of the phosphate regenerated relative to particulate phosphorus pools was an order of magnitude greater in Lake Michigan. Seventy percent of the nucleotidase activity was inhibited by 100 muM phosphate in the eutrophic lake, which suggests that most hydrolysis was by phosphatase. Therefore, nucleotidase activity may be more important to phosphorus regeneration in oligotrophic habitats than phosphatase activity.

Journal Article↗
PubMed1988

5'-Nucleotidase activity in the pineal organ of the pike. An electron-microscopic study.

To date, it is still unknown whether the metabolism of purine nucleotides and nucleosides plays an important role in the pineal organ of lower vertebrates. We have therefore investigated the sites of 5'-nucleotidase activity in the pineal organ of the pike (Esox lucius L.). Various ultracytochemical procedures were used. An intense ecto-5'-nucleotidase activity was characteristic of the entire plasma membrane of the phototransducers (cone-like and modified photoreceptor elements) and the interstitial cells, with exception of the portions facing the basal lamina of the pericapillary spaces. Additionally, intracellular sites of activity were also visualized in the inner segment and the pedicle of the phototransducers. Most of the intracellular deposits were apparently cytosolic and only few seemed to be associated with the membrane of the clear "synaptic" vesicles of the pedicle. Phagocytotic cells in the pineal lumen also showed a strong enzymatic activity on the outer surface of their plasmalemma (in ectoposition). This was apparently not the case for the cell types of the tissues surrounding the pineal vesicle. The present study emphasizes the importance of the occurrence and metabolism of purine nucleotides and nucleosides in a photoreceptive pineal organ.

5'-Nucleotidase↗
PubMed1989

Regional localization of human ecto-5' nucleotidase to chromosome 6q14-q21.

Human and mouse hybrids that contain fragments of human chromosome 6 as translocations were analysed for expression of ecto-5' nucleotidase enzymic activity measured by the conversion of AMP to adenosine and for antigenicity recognized by a monoclonal antibody specific for the human isozyme. Both methods allow a regional assignment of ecto-5'nucleotidase to 6q14-q21.

5'-Nucleotidase↗
PubMed1988

Isolation and characterization of bovine brain myelin distribution of 5'-nucleotidase.

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2',3'-cyclic nucleotide 3'-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5'-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH:cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5'-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.

5'-Nucleotidase↗
PubMed1982

Cytochemical heterogeneity of the glial plasma membrane: 5'-nucleotidase in retinal Müller cells.

Localization of 5'-nucleotidase in the retina of four rodent species was investigated using histochemical and cytochemical techniques. It was revealed by light microscopy that the enzyme is present in the receptor layer, the external nuclear layer and the external plexiform layer. It was found by electro microscopy that the glial cells of the Müller type have a strong activity on their plasmalemma in the enzyme-positive layers. With the exception of weak and scattered activity on some Müller cell processes forming horizontal fibres and the honeycomb meshwork in the ganglion cell layer, none of the internal layers contain 5'-nucleotidase. The asymmetric distribution of this ectoenzyme on the surface of the glial cells is a novel finding and demonstrates a surprising heterogeneity of glial plasmalemma.

5'-Nucleotidase↗
PubMed1988

A quantitative histochemical study of 5'-nucleotidase activity in rat liver using the lead salt method and polyvinyl alcohol.

5'-Nucleotidase (EC 3.1.3.5) activity was demonstrated in cryostat sections of rat liver using the Wachstein-Meisel medium and polyvinyl alcohol as tissue stabilizer. Optimum activity was obtained using an incubation medium containing 5 mM AMP, 10 mM magnesium chloride, 7.2 mM lead nitrate, 0.1 M Tris-maleate buffer, pH 7.2, and 17% (w/v) polyvinyl alcohol (Sigma, type III). The activity was localized at the bile canalicular and sinusoidal side of the plasma membranes of liver parenchymal cells as well as in the plasma membranes of endothelial cells of central veins and in fibroblasts surrounding portal tracts. The reaction was specific for 5'-nucleotidase because it was inhibited by ADP. Alkaline phosphatase did not interfere in the reaction. Cytophotometric analysis revealed a linear relationship between the formation of the final reaction product and incubation times up to 20 min and section thicknesses up to 8 micron. The activity in pericentral zones was 1.35 times the activity in periportal zones. The Michaelis constant for AMP was 1.4 mM in pericentral zones and 0.8 mM in periportal zones, suggesting that the bile canalicular and sinusoidal enzymes differ in their kinetic characteristics.

5'-Nucleotidase↗
PubMed1982

AMP deaminase, 5'-nucleotidase and adenosine deaminase in rat myocardial tissue in myocardial infarction and hypothermia.

AMP deaminase, 5'-nucleotidase and adenosine deaminase have been estimated in skeletal muscle and myocardial tissue in normal rats and in rats subjected to experimental myocardial infarction or hypothermia. A difference in the enzyme distribution was found between the right and left ventricles in the normal rat. A decrease in the activity of 5'-nucleotidase and an increase in the activity of adenosine deaminase were observed in infarcted myocardial tissue. The activity of all 3 enzymes was found to be depressed in the myocardium in rats subjected to hypothermia. These results are discussed in relation to adenosine production and its beneficial effects.

5'-Nucleotidase↗
PubMed1987

Effect of lecithin on the release of 5'-nucleotidase from liver plasma membrane of rat by bile acids.

Bile acids solubilize proteins from liver plasma membrane both in vivo and in vitro. The ability to solubilize the proteins is dependent on the species of bile acid. In this paper, the effect of phospholipid on the solubilization of a membrane-bound enzyme by bile acids was investigated in vitro. Taurocholate (TC) and tauroursodeoxycholate (TUDC) solubilized the enzyme, 5'-nucleotidase, from the liver plasma membrane of the rat in a concentration-dependent manner, although there was a great difference in their effect; at 40 mM, TC solubilized 55.4% of the original 5'-nucleotidase activity of the membrane, but TUDC only 5.7%. While lecithin alone had no solubilizing effect, its addition to the bile acids provoked a 10-fold increase in the solubilizing effect of TUDC, but virtually no change for TC, essentially equalizing the solubilizing effect of the two. Both TC-rich and TUDC-rich bile were obtained from rats infused with the respective bile acids via the jugular vein after their endogenous bile acid pool had been depleted. The solubilization effect of these biles was quite similar to the bile acid-lecithin mixtures. These findings demonstrate that lecithin enhances the ability of the bile acids to solubilize the membrane protein and eliminates the difference in the two bile acid species in their solubilizing ability.

5'-Nucleotidase↗
PubMedDate not supplied

Nitrobenzylthioinosinate binding sites in HeLa cells: relationship with ecto-5'-nucleotidase.

Nitrobenzylthioinosine (NBTI), a substrate for the ecto-5'-nucleotidase of HeLa cells, was used to probe the relationship between ecto-5'-nucleotidase dephosphorylation site and the dephosphorylated NBTI binding site. It may be assumed that the dephosphorylation site of the enzyme is separate and functions independently of the NBTI binding site. Evidences supporting these conclusions were based on the following observations: i. NBTI-P dephosphorylation progressed with similar rates in the presence or absence of NBTI in the incubation medium; ii. adenosine-5'-monophosphate inhibited the dephosphorylation of NBTI-P but did not affect the binding of NBTI. The inhibition was competitive and dependent on concentration; iii. the effect of the NBTI nonisotopic medium on the binding of free G-3H-NBTI and G-3H-NBTI derived from G-3H-NBTI-P was different, and an instantaneous isotopic dilution was observed with G-3H-NBTI, but not with G-3H-NBTI-P, as substrate.

5'-Nucleotidase↗
PubMed1981

Immunological evidence that plasma-membrane 5'-nucleotidase is a transmembrane protein.

Antibodies inhibiting specifically plasma membrane-bound 5'nucleotidase were used to determine the disposition of the enzyme in various lymphoma cell lines. Fluorescent Fab fragments of the inhibiting antibodies bound to MF2s and MOPC 173 plasmacytoma cells, whereas no fluorescence was observed with P 1798 thymoma cells in which the enzyme was absent. The relative potency of the antiserum in inhibiting the enzyme in right-side-out and inside-out plasma membrane vesicles prepared from the above cell lines indicated that various group of antigenic determinants were present. One group of antigenic determinants was present at the external face of the cell, and a second was associated with the inner surface of the membrane. A third group of antigenic determinants was located in the vicinity of the active site of the enzyme and it is the group that varied in the various plasmacytoma cells studied. The results are interpreted as immunological evidence that 5'nucleotidase is a transmembrane glycoprotein.

5'-Nucleotidase↗
PubMed1981

The effect of tris(hydroxymethyl) aminomethane on membrane bound 5'nucleotidase from swine aortic smooth muscle.

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity was stimulated by increasing the concentration of Tris over the range of 1--100 mM. Significant differences were found when the kinetic parameters of the enzyme determined in Tris were compared to those determined in glycine. These data suggest that Tris interacts with 5'-nucleotidase and is therefore unsuitable for use in the assay of the enzyme.

5'-Nucleotidase↗
PubMed1981

Regulation of cytosol 5'-nucleotidase by adenylate energy charge.

In the physiological range of the adenylate energy charge in liver (0.7-0.9), th rate of AMP-hydrolysis catalysed by rat liver cytosol 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) increased sharply with decreasing energy charge. In addition, a decrease in the concentration of Pi caused marked acceleration of the AMP-hydrolysing activity over the physiological range of adenylate energy charge. These responses seem to serve to protect the cells against a metabolic stress which could result from sudden utilization of ATP by removal of AMP. The AMP-hydrolysing activity of this enzyme decreased sharply as the size of the adenine nucleotide pool decreased in the physiological range. This effect may be a self-limiting response to prevent excess depletion of the pool. IMP-hydrolysing activity of this enzyme increased with increasing adenylate energy charge. But no marked response to its variation within the physiological range was observed. On the basis of the data obtained in this study, the IMP-hydrolysing activity of the cytosol 5'-nucleotidase in rat liver cells seems to be comparable to that of AMP deaminase reaction, but the AMP-hydrolysing activity was estimated to be less than 10% of AMP deaminase reaction at energy charge value of about 0.7. This strongly suggests that the AMP leads to IMP leads to inosine pathway is more significant that the AMP leads to adenosine leads to inosine pathway in rat liver.

5'-Nucleotidase↗
PubMed1981

Purification and some properties of cytosol 5'-nucleotidase from rat liver.

A 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was highly purified from rat liver. The preparation appeared homogeneous on the criteria of disc-gel electrophoresis. A pH optimum at about 6.5 was observed for all substrates tested. The activity of this enzyme was absolutely dependent on the presence of various bivalent metal salts. The highest V value was attained with MgCl2 and the concentration at half-enzyme saturation was lowest with MnCl2. The enzyme had markedly higher affinities for IMP, dIMP, GMP and dGMP than the other 5'-mononucleotides, although V values for all the substrates tested were in the same order of magnitude. The activity of this enzyme was stimulated by various alkali metal salts, some carboxylic acids and adenine nucleotides. When AMP was used as substrate, the substrate-velocity plot was sigmoidal and NaCl, Tris-maleate and ATP stimulated the enzyme by decreasing the sigmoidicity of the plot. When IMP was used as substrate, the substrate-velocity plot was hyperbolic and these three activators stimulated the enzyme by increasing the V and decreasing the Km value. Some of these results provided consistent evidence for the identity of this enzyme and the cytosol 5'-nucleotidase, the presence of which had been reported in crude preparations from rat liver.

5'-Nucleotidase↗
PubMed1982

A one-step determination of serum 5'-nucleotidase using a centrifugal analyzer.

We describe a one-step kinetic method for the determination of 5'-nucleotidase (EC 3.1.3.5). Inosine is formed by the hydrolysis of inosine 5'-monophosphate which is catalyzed by seric 5'-nucleotidase, and then is converted to hypoxanthine by nucleoside phosphorylase. Two moles of hydrogen peroxide are formed for each mole of hypoxanthine oxidized to urate by xanthine oxidase. The rate formation of hydrogen peroxide is monitored at 510 nm using the oxidation of the chromogenic system 3,5-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone in the presence of peroxidase. beta-Glycerophosphate inhibits the unspecific cleavage of the substrate by alkaline phosphatases. Inorganic phosphate is added to improve the reagent stability, and ferrocyanide to reduce bilirubin interference. Automation of the technique requiring 20 microliter of serum on a centrifugal analyzer is also described.

5'-Nucleotidase↗