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PubMedDate not supplied

[Post-irradiation changes in blood 5'-nucleotidase activity of rats after chronic exposure to gamma irradiation].

Kinetics of 5'-AMP hydrolysis by rat's blood serum was studied at 3, 10, 30, 90 and 180 days after prolonged gamma-irradiation at 0.1-1 Gy doses (60Co source, dose rate 4.4 x 10(-6) Gy/h). It was shown that more expressed kinetic anticooperativity of nucleotidase reaction in irradiated animals, as compare to control ones, was conditioned by composition predominance of low-affinity 5'-nucleotidases forms over their high-affinity patterns. The ratio of maximal velocities of high- to low-affinity 5'-nucleotidases was ranged from 2 in the control to 7 in irradiated rats at different postirradiation periods during the first 90 days of observation. It was suggested that appearance of new kinetic 5'-nucleotidases forms in blood stream of irradiated animals, predominantly with low-affinity, was attributed to impairment in the integrity of cellular membranes and may testify to postirradiation development of tissues injury.

5'-Nucleotidase↗
PubMed2006

Habituation to an open field alters ecto-nucleotidase activities in rat hippocampal synaptosomes.

ATP and adenosine may play a role in the mechanisms of synaptic plasticity and memory formation. Previous studies have shown that ecto-nucleotidase activities are altered during memory consolidation of an aversive task named step-down inhibitory avoidance. Here we investigate ecto-nucleotidase activities in hippocampal synaptosomes of rats submitted to training and test sessions of habituation to open field, which is one of the most elementary forms of learning. There were no significant alterations on ATP, ADP and AMP hydrolysis immediately after the training session. However, immediately after the test session (0min), there was a significant increase of ATP hydrolysis (61%), but not of ADP and AMP hydrolysis. Sixty minutes after the test session, a significant increase of NTPDase (75% and 60.5% for ATP and ADP hydrolysis, respectively) and ecto-5'-nucleotidase (40%) activities was observed. This study reveals the involvement of ecto-nucleotidase activities in different learning paradigms during memory processing.

Adenosine Triphosphatases↗
PubMed2003

Bacterial expression and characterization of a novel, soluble, calcium-binding, and calcium-activated human nucleotidase.

A newly discovered human analogue of a bed bug apyrase, which we named hSCAN-1 for human soluble calcium-activated nucleotidase-1, was expressed in bacteria, refolded from inclusion bodies, purified, and characterized. This apyrase, which is distinct from the eNTPDases exemplified by the endothelial CD39 (NTPDase1) apyrase, is a 38 kDa monomeric enzyme capable of hydrolyzing a variety of nucleoside di- and triphosphates, but not monophosphates. Preferred substrates include GDP, UDP, and IDP, with a pH optimum for activity between 6 and 7. The specific activity and substrate preference of the bacterially expressed enzyme closely mimic those of the enzyme expressed in mammalian COS cells, as well as the enzyme synthesized in an in vitro bacterial expression system. This suggests that glycosylation and other posttranslational modifications that do not occur in bacteria are not necessary for nucleotidase activity or proper folding of this human apyrase. hSCAN-1 absolutely requires Ca(2+), but not Mg(2+) or other divalent cations analyzed, for enzymatic activity. Surprisingly, the activity does not increase in a quasi-linear fashion at sub-millimolar Ca(2+) concentrations, as would be expected if Ca(2+) were only used as a cosubstrate for the nucleotide substrate, but rather follows a sigmoidal curve. The intrinsic fluorescence and difference absorption studies of hSCAN-1 in the absence of nucleotides revealed Ca(2+)-induced changes in the environment of tryptophan and tyrosine residues with half-saturation at about 90 microM Ca(2+). NaCl increased the half-saturating Ca(2+) concentration needed for both structural changes detected by optical spectroscopy and enzymatic activation of hSCAN-1 detected by nucleotidase assay. These results suggest that Ca(2+) triggers a conformational change in hSCAN-1, converting the enzymatically inactive protein to the active enzyme, in addition to forming the metal-nucleotide substrate complex necessary for nucleotidase activity.

Amino Acid Sequence↗
PubMed2001

Regulation of the ecto-nucleotidase pathway in rat hippocampal nerve terminals.

Ecto-nucleotidases play a pivotal role in terminating the signalling via ATP and in producing adenosine, a neuromodulator in the nervous system. We have now investigated the pattern of adenosine formation with different concentrations of extracellular ATP in rat hippocampal nerve terminals. It was found that adenosine formation is delayed with increasing concentrations of ATP. Also, the rate of adenosine formation increased sharply when the extracellular concentrations of ATP + ADP decrease below 5 microM, indicating that ATP/ADP feed-forwardly inhibit ecto-5'-nucleotidase allowing a burst-like formation of adenosine possibly designed to activate facilitatory A2A receptors. Initial rate measurements of ecto-5'-nucleotidase in hippocampal nerve terminals, using IMP as substrate, showed that ATP and ADP are competitive inhibitors (apparent Ki of 14 and 4 microM). In contrast, in hippocampal immunopurified cholinergic nerve terminals, a burst-like formation of adenosine is not apparent, suggesting that channelling processes may overcome the feed-forward inhibition of ecto-5'-nucleotidase, thus favouring A1 receptor activation.

Adenosine Triphosphatases↗
PubMed1998

Effects of lysophosphatidylcholine on the production of interstitial adenosine via protein kinase C-mediated activation of ecto-5'-nucleotidase.

1. Adenosine plays a crucial role in the evolution of ischemic preconditioning. With the use of microdialysis techniques in in situ rat hearts, we assessed the activity of ecto-5'-nucleotidase (a key enzyme responsible for adenosine production), and examined the effects of lysophosphatidylcholine (LPC) on the production of interstitial adenosine. 2. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rat hearts and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP, 100 microM). With this system, the dialysate adenosine originates from the dephosphorylation of AMP, catalyzed by endogenous ecto-5'-nucleotidase. The level of dialysate adenosine is a measure of the ecto-5'-nucleotidase activity in vivo. 3. LPC at concentrations of 25 and 50 microM significantly increased the level of dialysate adenosine to 122.7+/-4.3% (n=4, P<0.05) and 158.6+/-7.2% (n=5, P<0.05) of the control, respectively. Chelerythrine (200 microM), a protein kinase C (PKC) inhibitor, completely abolished the increase of dialysate adenosine afforded by LPC (50 microM) (n=5). 4. These data provide the first evidence that LPC does increase the concentration of interstitial adenosine in rat hearts in situ, through the PKC-mediated activation of endogenous ecto-5'-nucleotidase.

Adenosine↗
PubMed1981

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL.

Two forms (F-I and F-II) of 5'-nucleotidases (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N(6)-(Delta(2)-isopentenyl)adenosine 5'-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5'-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K(m) values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5'-phosphates, AMP being the preferred substrate. N(6)-(Delta(2)-isopentenyl)Adenosine 5'-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3'-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5'-nucleotidases may play an important role in regulating levels of "active cytokinin" in plant cells.

Journal Article↗
PubMed1995

Effects of P2-purinoceptor antagonists on ecto-nucleotidase activity of guinea-pig vas deferens cultured smooth muscle cells.

Ecto-nucleotidase activity was studied on primary cultures of guinea-pig vas deferens smooth muscle cells by measuring the inorganic phosphate (Pi) production using ATP as a substrate. The ecto-nucleotidase was insensitive to ouabain, oligomycin, sodium azide, p-nitrophenyl phosphate and B-glycerophosphate. Enzyme activity was highly dependent on either Ca2+ or Mg2+. Antagonists of P2X-purinoceptors, pyridoxalphosphate-6-azophenyl-2',4'- disulphonic acid, 4'4'-diisothiocyanatostilbene-2'2'- disulphonate, suramin and pyridoxal-5-phosphate, significantly inhibited ecto-nucleotidase activity. In contrast, the P1-purinoceptor antagonists, 8-p-sulphophenyl theophylline and 1,3-dipropyl-8-cyclopentylxanthine, did not affect the enzyme activity. Thus, when P2-purinoceptors are studied by testing agonists and antagonists potencies, the inhibition of ecto-nucleotidase activity by currently available P2-purinoceptor antagonists should be taken into account.

Adenosine Triphosphatases↗
PubMed1988

Cytophotometric analysis of alkaline phosphatase and 5'-nucleotidase activity in regenerating rat liver after partial hepatectomy.

Alkaline phosphatase and 5'-nucleotidase activities were analysed cytophotometrically in cryostat sections of female rat liver after partial hepatectomy. Alkaline phosphatase activity increased rapidly after operation up to a maximum seven-fold rise at 24 h in comparison with sham operated or control rats. There was no indication of preferential localization of alkaline phosphatase activity in either periportal or pericentral areas at any time point in control rats, sham operated rats or hepatectomized rats. Microscopical observation revealed that (a) all alkaline phosphatase activity was present at the bile canalicular surface of hepatocytes and (b) hepatocytes in mitosis did not show any increase in activity. These findings indicate that the high alkaline phosphatase activity after partial hepatectomy is not involved primarily in proliferation processes because cell division mainly takes place periportally. It may be needed for enhanced bile secretion by conversion of intracellular phosphorylcholine into choline which can be transported into the bile. The intracellular phosphorylcholine level is high after operation due to changes in phospholipid metabolism. 5'-Nucleotidase appeared to be three times higher pericentrally than periportally under normal conditions. Partial hepatectomy caused a 40 per cent decrease in activity in pericentral areas and only a small decrease periportally. It has been suggested that 5'-nucleotidase plays a role in breakdown of messenger RNA and its activity in control liver could be considerably lower periportally because plasma protein synthesis mainly takes place in this area.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1986

The distribution of adenosine A1 receptors and 5'-nucleotidase in the hippocampal formation of several mammalian species.

The distributions of adenosine A1 receptors, as demonstrated by 3H-cyclohexyladenosine (3H-CHA) binding, and the adenosine-producing enzyme 5'-nucleotidase were examined in the hippocampal formation of the rat, mouse, gerbil, cat, hamster, rabbit, and guinea pig. The enzyme and binding sites were restricted to subregions and often individual layers of this structure. The distribution of 3H-CHA binding was consistent among the species with the strata radiatum and oriens of fields CA1 and CA3 exhibiting the highest levels of binding. A distinct band of 3H-CHA binding was observed in the stratum moleculare of the dentate gyrus; and in most species, this band was restricted to the inner one-third of the stratum moleculare (i.e., proximal to the stratum granulosum). The strata pyramidale, granulosum, and lucidum were in general only weakly positive for 3H-CHA binding. The binding to the stratum lacunosum/moleculare (or the distinct strata lacunosum and moleculare in the rabbit and cat) was moderate. In contrast to the relative consistency of the patterns of 3H-CHA binding in these species, 5'-nucleotidase exhibited wide variations in both the absolute amount of activity and its localization. In all species, the strata granulosum and pyramidale appeared devoid of 5'-nucleotidase activity. The only clear exception to this rule was the CA3 region of the cat where activity was seen between the cell bodies of stratum pyramidale. The strata radiatum and oriens of CA1 were strongly positive in the rat and hamster but only low to moderately stained in the other species examined.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗
PubMed1984

Macrophage activation measured by changes in 5'-nucleotidase and alkaline phosphodiesterase I activities after infection of newborn and juvenile guinea pigs with mycobacterium microti.

Activation of alveolar, peritoneal, liver, and spleen macrophages was measured by decreased 5'-nucleotidase and elevated alkaline phosphodiesterase I activities in newborn and juvenile guinea pigs for up to 48 days after intracardiac or intranasal infection with Mycobacterium microti. Increased AP and reduced 5'-nucleotidase activity in cell lysates of macrophages appeared to be related to the carrier state during infection. Intranasal infection of 8-week-old guinea pigs with M. microti produced decreases of 5'-nucleotidase activity in alveolar macrophages during 1-8 weeks post infection and in macrophages from all four sources from 4-8 weeks.

5'-Nucleotidase↗
PubMed1980

Effect of ethanol on neural cells grown in culture: interaction with plasma membrane ecto-5'-nucleotidase activity.

The effect of ethanol on an enzyme system within an intact functional plasma membrane has been studied using neural cells grown in culture. Rat C6 glioma cells in mono-layer culture were treated acutely or chronically with 100 mM ethanol and the effect of this exposure on the activity of ecto-5'-nucleotidase was determined. Acute exposure led to an increase in enzyme activity with maximum stimulation occurring at concentrations of 100 - 400 mM ethanol. Chronic treatment of cells with 100 mM ethanol for 4 - 8 days also caused an increase in ecto-5'-nucleotidase activity. Both the acute and chronic ethanol-induced stimulation of enzyme activity was completely reversible by removing the ethanol; the acute effects reversed immediately, whereas the chronic effects required several hours. The addition of Concanavalin A demonstrated that the effects on enzyme activity of both chronic and acute exposure to ethanol were blocked by modification of the external cell surface. The effect of chronic exposure to 100 mM ethanol was further localized to an action on the plasma membrane by studies which showed chronic exposure to have no effect on the intracellular 5'-nucleotidase activity. Furthermore, the occurrence of pharmacological tolerance to acute ethanol was observed in this plasma membrane system following chronic treatment of C6 cells with 100 mM ethanol. These findings are consistent with the hypothesis that mammalian neural cells can adapt to the chronic presence of ethanol through changes in their plasma membrane.

5'-Nucleotidase↗
PubMed1986

Electron microscopic localization of 5'-nucleotidase in rat salivary glands. Comparative enzyme- and immunohistochemical studies.

The localization of 5'-nucleotidase in rat parotid and submandibular glands was investigated at the electron microscope level by an immunohistochemical technique using a highly specific antibody, and the results were compared with those obtained using the newley developed cerium method for enzyme histochemistry. Both methods demonstrated that 5'-nucleotidase is located on the external surface of the luminal plasma membranes of acinar cells as well as on intercalated and striated ductal cells. In the basolateral membranes of these cells, the portions adjacent to myoepithelial cells exhibited intense reaction products, but the other areas of plasma membranes contained only trace amounts of the reaction products. Both cerium-based enzyme histochemistry and immunohistochemistry showed that myoepithelial cells retain the enzyme on their plasma membranes. Neither method produced reaction products in the intracytoplasmic structure of constitutive cells of the salivary glands. We discuss the usefulness of the cerium-ion method for the demonstration of 5'-nucleotidase activity and compare it with the traditional lead-ion method.

5'-Nucleotidase↗
PubMed1982

A cytochemical method for the demonstration of 5'-nucleotidase in mouse peritoneal macrophages, with cerium ions used as trapping agent.

A method was developed for the demonstration of 5'-nucleotidase in murine peritoneal resident macrophages. The cells are incubated cytochemically without agitation and cerium chloride is used as a trapping agent. Under these conditions, the great majority of the macrophages in the unstimulated peritoneal cavity show enzyme activity in the plasma membrane. In the presence of AMP-S (an AMP analogue inhibiting 5'-nucleotidase, as shown biochemically) there was a decrease in both the number of positive macrophages and the amount of reaction product on the plasma membranes. This indicates that the enzyme activity detected by our cytochemical procedure is attributable to 5'-nucleotidase.

5'-Nucleotidase↗
PubMed1983

5'-deoxy-5'-thioanalogs of adenosine and inosine 5'-monophosphate: studies with 5'-nucleotidase and alkaline phosphatase.

The interaction of 5'-deoxy-5'-thioadenosine 5'-monophosphate (A(S)MP) and 5'-deoxy-5'-thioinosine 5'-monophosphate (I(S)MP) with snake venom, 5'nucleotidase, and calf intestinal mucosa alkaline phosphatase has been characterized. The substrates, A(S)MP and I(S)MP, are analogs of adenosine 5'-monophosphate and inosine 5'-monophosphate in which sulfur replaces oxygen as the bridge between the 5'-carbon of the ribose and the phosphorous. The P-S bond of both A(S)MP and I(S)MP was hydrolyzed by alkaline phosphatase producing the corresponding thionucleoside as a reaction product. The Km for A(S)MP was 270 microM and the V for alkaline phosphatase was 110 nmol/min/mg (8% of the V for AMP), whereas the corresponding values for I(S)MP were 300 microM and 530 nmol/min/mg protein, respectively. In contrast, 5'-nucleotidase did not catalyze hydrolysis of either A(S)MP or I(S)MP. A(S)MP and I(S)MP were competitive inhibitors of the 5'-nucleotidase hydrolysis of AMP and IMP, respectively, with Ki values of 975 and 13 microM. Decreasing the pH of the reaction from 8.1 to 7.1 lowered the Ki for I(S)MP by 100-fold, to a value of 0.15 microM.

5'-Nucleotidase↗
PubMed1981

Solubilization of rat heart sarcolemma 5'-nucleotidase by phosphatidylinositol-specific phospholipase C.

The influence of a phosphatidylinositol-specific phospholipase C treatment on rat heart sarcolemmal 5'-nucleotidase was investigated. Upon complete hydrolysis of all phosphatidylinositol in the sarcolemma, 75% of 5'-nucleotidase activity was found in the solubilized form. The insolubilized enzyme after this treatment has the same Km for AMP as the untreated, sarcolemmal-bound enzyme (0.04 mM), whereas the solubilized enzyme has a 40-fold increase in Km for AMP (0.16 mM). Other sarcolemmal-bound enzymes were not affected by the same treatment. Hence, the specific involvement of phosphatidylinositol in the binding of 5'-nucleotidase to the sarcolemma of the rat heart is clearly demonstrated.

5'-Nucleotidase↗
PubMed1986

Diadenosine tetraphosphate activates cytosol 5'-nucleotidase.

The rate of hydrolysis of IMP (0.5 mM) by cytosol 5'-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5'-nucleotidase from rat liver.

5'-Nucleotidase↗
PubMed1982

Effect of chronic alcohol consumption on the activities of liver plasma membrane enzymes: gamma-glutamyltransferase, alkaline phosphatase and 5'-nucleotidase.

To study the effect of chronic alcohol administration on the activities of liver plasma membrane enzymes such as gamma-glutamyltransferase, alkaline phosphatase and 5'-nucleotidase, female rats were pair-fed for 6 weeks nutritionally adequate liquid diets containing either ethanol or isocaloric carbohydrates as controls. Compared to the control diet, chronic alcohol administration resulted in a significant enhancement of serum activities of gamma-glutamyltransferase, alkaline phosphatase and 5'-nucleotidase by 91% (P less than 0.005), 80% (P less than 0.001) and 65% (P less than 0.01), respectively. Concomitantly, chronic alcohol intake led to a striking increase of gamma-glutamyltransferase activities in liver homogenates by 68% (P less than 0.001), in liver plasma membranes rich in bile canaliculi by 80% (P less than 0.025), and in liver plasma membranes free of bile canaliculi by 24% (P less than 0.02). However, chronic ethanol consumption had no effect on alkaline phosphatase activities in liver homogenates and liver plasma membranes but significantly suppressed 5'-nucleotidase activities. These results therefore show that chronic intake of ethanol increases serum activities of enzymes originating from liver plasma membranes but has different effects on the enzyme activity in liver plasma membranes itself, suggesting that the alcohol-mediated increase of serum activities of various enzymes originating from liver plasma membranes might be due to different mechanisms.

5'-Nucleotidase↗
PubMed1988

Polyclonal and monoclonal antibodies against chicken gizzard 5'-nucleotidase inhibit the spreading process of chicken embryonic fibroblasts on laminin substratum.

Polyclonal and monoclonal antibodies raised against chicken gizzard 5'-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5'-nucleotidase. However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard. In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5'-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.

5'-Nucleotidase↗