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At least 703 records · Page 39Linked to original sources
PubMed1991

Differential theophylline inhibition of alkaline phosphatase and 5'-nucleotidase of bovine milk fat globule membranes.

1. The effects of theophylline (1,3-dimethylxanthine) on alkaline phosphatase and 5'-nucleotidase activities of bovine milk fat globule membranes (MFGM) were examined. 2. Theophylline inhibited MFGM alkaline phosphatase in a concentration-dependent manner with 50% inhibition produced by 99 +/- 28 microM theophylline. 3. The 5'-nucleotidase activity was resistant to theophylline inhibition with 50% inhibition produced by 33.9 +/- 3.1 mM theophylline. 4. Theophylline was an uncompetitive inhibitor of MFGM alkaline phosphatase with a Ki of 126 +/- 15 microM. 5. The extent of theophylline inhibition of alkaline phosphatase activity was independent of the substrate utilized in the assay. 6. The effect of theophylline on bovine MFGM alkaline phosphatase was similar to theophylline effects on other mammalian alkaline phosphatases of liver/bone isoenzyme origin.

5'-Nucleotidase↗
PubMed1993

Altered properties of human T-lymphoblast soluble low Km 5'-nucleotidase: comparison with B-lymphoblast enzyme.

Soluble low Km 5'-nucleotidases have been purified from human cultured T- and B-lymphoblasts to compare their properties and to examine the mechanism of different rates of nucleotide dephosphorylation. The enzyme from B-lymphoblasts (MGL-8) was 4385-fold purified with a specific activity of 114 mumol/min/mg, while the enzyme from T-lymphoblasts (CEM, MOLT-4) was 4355-fold purified with a specific activity of 35 mumol/min/mg. The activity of both enzymes have an absolute requirement for Mg++. The B-cell enzyme has maximum activity with Mg2+ > Mn2+ > Co2+, while the T-cell enzyme had maximum activity with Co2+ > Mn2+ > Mg2+. The optimum activity was at pH 7.4-9.0 for the B-cell enzyme and pH 9.0 for the T-cell enzyme. Substrate specificity was the same for both enzymes with the following relative Vmax values: CMP > UMP > dUMP > dCMP > dAMP > IMP > GMP > dIMP > dGMP. The Km values for AMP and IMP were 12 and 25 microM for the B-cell enzyme, and 7.0 and 12 microM for the T-cell enzyme. ATP and ADP are competitive inhibitors of these enzymes with apparent Ki values of 100 and 20 microM for the B-cell enzyme, and 44 microM and 8 microM for the T-cell enzyme, respectively. The apparent molecular mass by gel filtration column chromatography is 145 kD for the B-cell enzyme and 72 kDa for the T-cell enzyme. The subunit molecular masses by Western blots are 69.2 kD for both enzymes. These properties suggest that the B-lymphoblast enzyme is identical or similar to the enzyme from human placenta. However, the T-cell enzyme has some different properties. We conclude that these differences plus a lower content of low Km 5'-nucleotidase in T-cells may account for the decreased ability of T-lymphoblasts to dephosphorylate nucleotides and may contribute to the selective cytotoxicity of deoxyribonucleosides for T-lymphoblasts as compared to B-lymphoblasts.

5'-Nucleotidase↗
PubMed1992

Structural and functional relationships in 5'-nucleotidase from bull seminal plasma. A Fourier transform infrared study.

Fourier transform infrared spectroscopy (FTIR) was used to investigate the secondary structure of 5'-nucleotidase from bull seminal plasma (BSP). Spectra of protein in both D2O and H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide I band. The protein, which is made up of two apparently identical subunits and which contains two zinc atoms, was studied in its native form, in the presence of dithiotreitol (DTT) and after removal of the two zinc atoms by means of nitrilotriacetic acid (NTA). Deconvolved and second derivative spectra of amide I band showed that the native protein contains mostly beta-sheet structure with a minor content of alpha-helix. The quantitative analysis of the amide I components was performed by a curve-fitting procedure which revealed 54% beta-sheet, 18% alpha-helix, 22% beta-turns and 6% unordered structure. The second derivative and deconvolved spectra of amide I band showed that no remarkable changes in the secondary structure of 5'-nucleotidase were induced by either DTT or NTA. These results were confirmed by the curve-fitting analysis where little or no changes occurred in the relative content of amide I components when the protein was treated with DTT or with NTA. Major changes, however, were observed in the thermal denaturation behavior of the protein. The native protein showed denaturation at temperatures between 70 and 75 degrees C, while the maximum of denaturation was observed between 65 and 70 degrees C and between 55 and 60 degrees C in the presence of NTA and DTT, respectively. The results obtained indicate that the two separate subunits of the protein have essentially the same secondary structure as that of the native enzyme.

5'-Nucleotidase↗
PubMed1993

Characterization of a filtrable 3'-nucleotidase of Leishmania donovani.

A plasma membrane 3'-nucleotidase (3'-ribonucleotide phosphohydrolase, EC 3.1.3.6) having a apparent subunit molecular mass of 38 kDa but filtrable through a Centriprep-10 microconcentrator (Amicon) membrane was purified from Leishmania donovani promastigotes. The enzyme activity has an optimum at pH around 7.5. EDTA strongly inhibited the enzyme activity which was fully restored only by Co2+, from various metal ions added. Citrate ions, Zn2+ or dithiothreitol were also strongly inhibitory. The enzyme is apparently located on the inner side of the parasite plasma membrane. The substrate specificity and kinetics of the filtrable enzyme are similar to those of the non-filtrable outer-surface-membrane-bound 3'-nucleotidase reported by Gbenle and Dwyer (Gbenle, G.O. and Dwyer, D.M. (1992) Biochem. J. 285, 41-46). Therefore, it is suggested that both enzymes are implicated in the supply of nucleosides to the parasite.

Animals↗
PubMed1987

Renaturation of Leishmania donovani 3'-nucleotidase following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. Renaturation of a 3'-nucleotidase from the surface membrane of Leishmania donovani promastigotes was achieved following polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). 2. Enzyme activity was detected in situ in gels, following SDS removal, by incubating the gels in reaction mixtures containing 3'-AMP or 3'-UMP as substrate followed by staining for the inorganic phosphate (Pi) reaction product with malachite green-molybic acid solution. 3. Conditions for the removal of SDS by diffusion and for the renaturation of enzyme activity are described including evidence for the detergent requirement, which is best satisfied by 3[(3-cholamidopropyl)-dimethylammonio]2-hydroxy-1-propane sulfonate (CHAPSO). 4. Results indicate that the 3'-nucleotidase migrates under these conditions as a polypeptide with an Mr of 43,000.

Animals↗
PubMed1990

Dependence between 5'-nucleotidase activity and phosphatidylcholine content in pig adipocyte plasma membrane. Absence of relationship with membrane physical state.

1. 5'-Nucleotidase activity has been examined in pig adipocyte plasma membrane, in two different adipose sites. 2. Membrane fluidity has been investigated using 1,6-diphenylhexatriene (DPH). 3. In this work, evidence was found for a tissue specificity of 5'-nucleotidase (E.C. 3.1.3.5) activity. 4. The enzyme activity was highly correlated to the membrane phosphatidylcholine content. 5. The latter parameters remained unmodified when large alterations in membrane composition and physical state were produced by means of the dietary n-6 PUFA content manipulations.

5'-Nucleotidase↗
PubMed1992

A comparative study on tissue distribution and metabolic adaptation of IMP-GMP 5'-nucleotidase.

1. Activity of a cytoplasmic 5'-nucleotidase which preferentially hydrolyzes IMP and GMP (IMP-GMP 5'-nucleotidase) was determined by a specific immunochemical method in two species of birds and two species of mammals. 2. The activity was markedly high in avian liver, and it increased two-fold in response to a high protein diet in chicken liver. 3. In mammals, the activity was high in testis and spleen. In the rat, the activities in liver, kidney and heart extracts increased by about 30% in response to the high protein diet, while they increased three-fold in regenerating liver. 4. Low activities were detected in skeletal muscles and in erythrocytes of all the species studied.

5'-Nucleotidase↗
PubMed2005

IMP-GMP 5'-nucleotidase in reptiles: occurrence and tissue distribution in a crocodile and three species of lizards.

IMP-hydrolyzing activity (which is reactive with goose anti-pig lung IMP-GMP 5'-nucleotidase (c-N-II: EC.3.1.3.5) serum) was detected in extracts from several tissues (liver, heart, kidney, spleen, stomach, lung and skeletal muscle) from constitutively uricotelic reptiles: a crocodile (Crocodylus siamensis), and three species of lizard (Furcifer oustaleti, Tupinambis rufescens and Varanus gouldi). The activities were markedly high in the livers: 3.0 units/g in the crocodile and 1.4-2.9 units/g in the lizards. These were similar to those previously reported for the livers from chicken and snakes (also constitutively uricotelic), and 4- to 10-fold higher than those in ammoniotelic or ureotelic vertebrates. These findings suggest that the high activity of IMP-GMP 5'-nucleotidase in the liver is a feature of constitutive uricotelism, and that the enzyme may participate in the production of uric acid as an end product of amino acid catabolism.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

NTPDase and 5'-nucleotidase activities in rats with alloxan-induced diabetes.

Diabetes is associated with a hypercoagulable state. In this study, we investigated the potential effects of alloxan-induced diabetes on the activities of the enzymes NTPDase (E.C. 3.6.1.5, apyrase, ATP diphosphohydrolase, ecto/CD39) and 5'-nucleotidase (E.C. 3.1.3.5, CD73) that can control the levels of ADP and adenosine, two substances that regulates platelet aggregation. In the alloxan-treated rats, NTPDase activity was significantly increased by 88 and 35% with ATP as substrate and by 156 and 58% with ADP as substrate in platelets and synaptosomes, respectively (P< 0.05). AMP hydrolysis was increased by 142% (platelets) and 70% (synaptosomes) in diabetic rats compared to control. These results demonstrate that alloxan-induced diabetes interferes with ATP, ADP, and AMP hydrolysis in platelets and synaptosomes. Taken together, these results may indicate that in diabetic rats both NTPDase and 5'-nuleotidase from the central nervous system (CNS) and platelets respond similarly with increased activity. Thus, we speculate that platelets could be used as a potential peripheral marker of central alterations in NTPDase and 5'-nucleotidase activities in diabetes.

5'-Nucleotidase↗
PubMed2003

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

The enzymatic properties of an ecto-5'-nucleotidase were described in Trichomonas vaginalis. The enzyme hydrolyzes nucleoside monophosphates in optimum pH values of 7.5 and 6.5 for the 30236 strain and for the 30238 strain, respectively. Mg(2+) and Ca(2+) were activators of AMP hydrolysis in both strains. The apparent K(m) (Michaelis constant) values for Mg(2+)-AMP were 111.4+/-28.1 microM (mean+/-SD, n=3) for 30236 strain and 420.2+/-35.7 microM (mean+/-SD, n=3) for 30238 strain. The ecto-5'-nucleotidase activity was insensitive to levamisole and tetramisole, inhibitors of alkaline phosphatases, whereas alpha,beta-methylene-ADP inhibited the enzymatic activity of both strains. Our results showed that the AMP hydrolysis presents differences in some kinetic parameters between the two strains investigated. An analysis of the enzymatic chain involved in the ATP hydrolysis to adenosine will contribute to understanding the biochemical aspects of the parasite and the mechanisms related to host-parasite interactions.

5'-Nucleotidase↗
PubMed2005

Inhibition of NTPDase, 5'-nucleotidase, Na+/K+-ATPase and acetylcholinesterase activities by subchronic treatment with Casearia sylvestris.

The aqueous extract of Casearia sylvestris was tested in cortical membrane preparations. C. sylvestris was obtained commercially from two different sources, designated as Sample A and Sample B. The enzymes studied in this work were NTPDase-like, 5'-Nucleotidase, Na(+)/K(+)-ATPase and acetylcholinesterase (AChE). Adult rats received aqueous extracts from C. sylvestris in a dose of 20mg/kg body wt. daily for a 75-day-period, by oral administration (gavage). Our study showed that this treatment caused an inhibition of NTPDase-like activity with both, ATP (19.41% with Sample A and 25.03% with Sample B) and ADP (41.57% with Sample A and 31.20% with Sample B) as substrates. This treatment also caused an inhibition of 5'-nucleotidase activity (28.34% with Sample A and 31.46% with Sample B) and Na(+)/K(+)-ATPase (25.08% with Sample A and 24.81% with Sample B). The rate of acetylcholine degradation was reduced, as shown by the inhibition of AChE (31.65% and 26.74%, Samples A and B, respectively). These results suggest that extracts of C. sylvestris can cause neurochemical alterations in the purinergic and cholinergic systems of the central nervous system.

5'-Nucleotidase↗
PubMed2000

Ecto-nucleotidase of cultured rat superior cervical ganglia: dipyridamole is a novel inhibitor.

Based on studies of agonist potencies on intact rat superior cervical ganglia, it has been suggested that this ganglion possesses distinct receptors for purine and pyrimidine nucleotides. However, the potency of an agonist is dependent upon whether it is susceptible to extracellular metabolism by the tissue. The aim of this investigation was to study the metabolism of uridine or adenosine nucleotides and nucleosides and the effects of dipyridamole and an ecto-ATPase inhibitor ARL 67156 (6-N, N-diethyl-D-beta-gamma-dibromomethylene-ATP) on their metabolism. Adenosine- and uridine-5'-triphosphates (ATP and UTP) were catabolised by cultured rat superior cervical ganglia, to their di- and monophosphates. Both ATP and UTP breakdown was significantly inhibited by dipyridamole (10 mcM), whereas ARL 67156 (100 mcM), was a weaker inhibitor of ATP degradation and inhibited UTP breakdown by approximately 40%. Metabolism of ATP and UTP by cultured rat superior cervical ganglia was reduced after treatment with cytosine-beta-arabinoside, suggesting that non-neuronal cells along with neuronal cells contribute to their breakdown. In conclusion, these results indicate that rat superior cervical ganglia possess ecto-nucleotidases capable of catabolising purine and pyrimidine nucleotides to their nucleosides, and that dipyridamole is a potent inhibitor of ecto-nucleotidase activity.

5'-Nucleotidase↗
PubMed2001

Cytomegalovirus infection increases the expression and activity of ecto-ATPase (CD39) and ecto-5'nucleotidase (CD73) on endothelial cells.

We describe enhanced expression and enzymatic activity of ecto-ATPase and ecto-5'nucleotidase on CMV infected endothelial cells as compared to uninfected cells. These ectoenzymes play a major role in modulation of platelet activation and aggregation. Furthermore, adenosine has a modulatory effect upon inflammation. Addition of ATP, ADP or AMP to cultures of CMV infected or uninfected endothelial cells revealed increased turnover of AMP in CMV infected endothelial cells. In addition, the superoxide production by stimulated polymorphonuclear cells was inhibited in the presence of CMV infected endothelial cells as compared to uninfected cells, probably due to the enhanced activity of ecto-5'nucleotidase and associated to production of adenosine.

5'-Nucleotidase↗
PubMed1997

Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides.

Two cytoplasmic forms of pyrimidine nucleotidase (PN-I and PN-II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates respectively. PN-I and PN-II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3'-azido-3'-deoxy-thymidine (AZT), cytosine-beta-D-arabinofuranoside (AraC) and 5-fluoro-2'-deoxy-uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN-I and PN-II. PN-I phosphotransferase activity revealed higher affinity for oxynucleosides with respect to deoxy-nucleosides, whereas the contrary seems to be true for PN-II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine-specific phosphotransferase activity.

Erythrocytes↗
PubMed1999

Human monocyte derived dendritic cells express functional P2X and P2Y receptors as well as ecto-nucleotidases.

We investigated the expression and function of P2 receptors and ecto-nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT-PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA-2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor-alpha, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto-apyrase CD39 and the ecto-5'-nucleotidase CD73 as demonstrated by RT-PCR. Extracellular ATP was rapidly hydrolyzed by these ecto-enzymes as shown by separation of 3H-labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.

5'-Nucleotidase↗
PubMed1977

Lymphocyte purine 5'-nucleotidase edficiency in primary hypogammaglobulinaemia.

The mean activity of purine 5'-nucleotidase (E.C.3.1.3.5) in lymphocytes was significantly lower in patients with non-familial adult onset "variable" primary hypogammaglobulinaemia than in non-hypogammaglobulinaemic control subjects. These findings suggest that 5'-nucleotidase activity is necessary for normal lymphocyte function. This may be related to its role in facilitating the transfer of newly formed purines across cell membranes by converting them from nucleotides to nucleosides. This enzyme defect may be a characteristic metabolic lesion in at least some of the patients; alternatively, it may be secondary to some other metabolic defect or plasma-membrane change.

Agammaglobulinemia↗
PubMed2001

Cloning of a mouse cytosolic 5'-nucleotidase-I identifies a new gene related to human autoimmune infertility-related protein.

Adenosine production catalysed by cytosolic 5'-nucleotidase (cN-I) regulates diverse physiological processes. We report here a mouse cN-I (mcN-I) cloned from heart and testis. The open reading frame contains several potential translation initiation sites, which yield similarly active 5'-nucleotidases. Using overexpression in COS-7 cells we showed that mcN-I, like the previously cloned pigeon cN-I, is activated by ADP and catalyses adenosine formation during ATP breakdown. The N- and C-termini of mcN-I and pcN-I are divergent. Deletion of the 12 C-terminal amino acids or the first 19 N-terminal amino acids of pcN-I does not diminish activity, although deletion of the first 31 N-terminal amino acids reduces activity by 70%. Overall mcN-I is only 66% identical to pcN-I or the recently cloned human cN-I (hcN-I), while hcN-I and pcN-I are 85% identical. We report here a partial hcN-I sequence that is only 70% identical with the published hcN-I amino acid sequence but is 87% identical with mcN-I. Both hcN-I sequences have perfect matches to distinct human genome sequences. Our data imply the existence of at least two genes for cN-I, cN-I(A), previously cloned from pigeon and human, and cN-I(B) that we report here from mouse and partially from human.

5'-Nucleotidase↗
PubMed2003

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis.

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.

5'-Nucleotidase↗