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PubMed1998

IMP-GMP 5'-nucleotidase from rat brain: activation by polyphosphates.

IMP-GMP 5'-nucleotidase has been purified to homogeneity from total rat brain extracts. This preparation showed a unique band (Mr 54,000 +/- 1,509) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme presented the following properties: optimal pH value, 6.5-6.8; relative velocity measured in the presence of MgCl2, MnCL2, CoCl2, and NiCl2 (2 mM), 100, 60, 11, and <1, respectively; preferred substrates, IMP and GMP; and activation constant (Ka) found for Ap4A, Ap5A, and Ap6A, 83 +/- 38, 77 +/- 32, and 57 +/- 12 microM, respectively. Under assay conditions where activation by Ap4A was fivefold, the activation produced by dinucleotides was as follows: Ap4G (4.0), Ap4I (2.9), Ap4X (3.3), Ap4C (0.7), Ap4U (1.1), Ap4epsilonA (1.5), Ap4ddA (1.7), Gp4G (2.2), Ap3A (1.1), and Ap2A (1.2). Polyphosphates P18, P19, P20, and P35 were activators of the reaction with calculated Ka values of 3.5 +/- 0.5, 0.9 +/- 0.2, 0.6 +/- 0.2, and 1.3 +/- 0.5 microM, respectively. The following compounds, at 0.1 mM, were effectors of the phosphotransferase reaction producing the fold activation indicated: Ap4A (8.3), Ap5A (10.2), Ap6A (10.1), Ap4G (7.7), Ap4X (7.6), Ap4U (2.1), glycerate 2,3-bisphosphate (3.9), and unpurified P15 (7.6). Two enzyme forms of IMP-GMP 5'-nucleotidase were detected when the extracts from rat tissues or from the crustacean Artemia were subjected to chromatography on a Dyematrex Green A column. The ratio of the hydrolytic activities under both peaks (peak I/peak II) was as follows: brain (1.5), heart (1.9), liver (1.6), lung (2.0), testis (3.8), and Artemia cysts (2.0).

5'-Nucleotidase↗
PubMed1991

Effect of castration and anatomical site on the plasma membrane structure and the activities of adenylate cyclase and 5'-nucleotidase in pig adipocytes.

The effects of castration on fat cell plasma membrane structure and enzyme activities (adenylate cyclase and 5'-nucleotidase) were studied in pig adipose tissues in two fat deposits (subcutaneous and perirenal). Castration induced a fat cell enlargement in both tissues. Membrane cholesterol content was reduced and fluidity was increased in perirenal fat from castrated animals. Castration had no effect on 5'-nucleotidase activity which was higher in subcutaneous than in perirenal in both kinds of animals. Adenylate cyclase activity was studied in the presence of different effectors: isoproterenol-stimulations of the enzyme were not affected by castration but were site-specific. GppNHp-stimulated activities were increased in subcutaneous fat from castrated animals. Castration had no influence on forskolin stimulations. The magnitude of GppNHp- and forskolin-stimulated activities were found to be tissue-dependent. Membrane results are discussed in relation with castration-induced fat cell enlargement.

5'-Nucleotidase↗
PubMed1979

Biochemical basis for differential deoxyadenosine toxicity to T and B lymphoblasts: role for 5'-nucleotidase.

Deoxyadenosine metabolism was investigated in cultured human cells to elucidate the biochemical basis for the sensitivity of T lymphoblasts and the resistance of B lymphoblasts to deoxyadenosine toxicity. T lymphoblasts have a 20-to 45-fold greater capacity to synthesize deoxyadenosine nucleotides than B lymphoblasts at deoxyadenosine concentrations of 50--300 micron. During the synthesis of dATP, T lymphoblasts accumulate large quantities of dADP, whereas B lymphoblasts do not accumulate dADP. Enzymes affecting deoxyadenosine nucleotide synthesis were assayed in these cells. No substantial differences were evident in activities of deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76) or deoxyadenylate kinase [ATP:(d)AMP phosphotransferase, EC 2.7.4.11]. The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was increased 44-fold for AMP and 7-fold for dAMP in B lymphoblasts. A model for the regulation of deoxyadenosine nucleotide synthesis by 5'-nucleotidase activity is proposed on the basis of the observations.

Adenosine Deaminase↗
PubMed2000

Human cytosolic 5'-nucleotidase I: characterization and role in nucleoside analog resistance.

Nucleoside analogs are important in the treatment of hematologic malignancies, solid tumors, and viral infections. Their metabolism to the triphosphate form is central to their chemotherapeutic efficacy. Although the nucleoside kinases responsible for the phosphorylation of these compounds have been well described, the nucleotidases that may mediate drug resistance through dephosphorylation remain obscure. We have cloned and characterized a novel human cytosolic 5'-nucleotidase (cN-I) that potentially may have an important role in nucleoside analog metabolism. It is expressed at a high level in skeletal and heart muscle, at an intermediate level in pancreas and brain, and at a low level in kidney, testis, and uterus. The recombinant cN-I showed high affinity toward dCMP and lower affinity toward AMP and IMP. ADP was necessary for maximal catalytic activity. Expression of cN-I in Jurkat and HEK 293 cells conferred resistance to 2-chloro-2'-deoxyadenosine, with a 49-fold increase in the IC(50) in HEK 293 and a greater than 400-fold increase in the IC(50) in Jurkat cells. Expression of cN-I also conferred a 22-fold increase in the IC(50) to 2',3'-difluorodeoxycytidine in HEK 293 cells and an 82-fold increase in the IC(50) to 2',3'-dideoxycytidine in Jurkat cells. These data indicate that cN-I may play an important role in the regulation of physiological pyrimidine nucleotide pools and may also alter the therapeutic efficacy of certain nucleoside analogs.

5'-Nucleotidase↗
PubMed2001

Bovine cytosolic 5'-nucleotidase acts through the formation of an aspartate 52-phosphoenzyme intermediate.

Cytosolic 5'-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5'-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5'-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles.

5'-Nucleotidase↗
PubMed2003

Ecto 5'-nucleotidase and nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways.

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.

5'-Nucleotidase↗
PubMed2004

Triatoma infestans apyrases belong to the 5'-nucleotidase family.

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.

5'-Nucleotidase↗
PubMed2006

Hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency: a biochemical, genetic and clinical overview.

Pyrimidine 5' -nucleotidase (P5'N-1) deficiency is the third most common enzyme abnormality after glucose 6-phosphate dehydrogenase and pyruvate kinase causing hereditary non-spherocytic hemolytic anemia. The disease is transmitted as an autosomal recessive trait. The degree of hemolysis is generally mild-to moderate. The hallmark of this enzyme deficiency is the presence of pronounced basophylic stippling in red blood cell peripheral blood smear together with accumulation of pyrimidine nucleotides within erythrocytes. No correlation has been found between residual activity and degree of hemolysis. The structural human gene for P5'N-1 is now available and fifteen different mutations had been identified so far. More recently, a functional analysis of P5'N-1 mutants had been performed providing a rationale for the pathological effects of the mutations. All mutations investigated affect amino acid residues unambiguously essential for the catalytic efficiency and/or protein stability, suggesting drastic reduction of the enzyme activity in red blood cells of patients affected by the disorder. Nevertheless, some patients exhibit high residual P5'N-1 activity, suggesting that P5'N-1 deficiency is compensate by other nucleotidases and/or alternative pathways in nucleotide metabolism. No specific therapy for P5'N-1 deficiency is now available.

5'-Nucleotidase↗
PubMed2006

F-ara-AMP is a substrate of cytoplasmic 5'-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation.

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5'-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5'-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.

5'-Nucleotidase↗
PubMed2004

Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia.

Extracellular adenosine has been widely implicated in adaptive responses to hypoxia. The generation of extracellular adenosine involves phosphohydrolysis of adenine nucleotide intermediates, and is regulated by the terminal enzymatic step catalyzed by ecto-5'-nucleotidase (CD73). Guided by previous work indicating that hypoxia-induced vascular leakage is, at least in part, controlled by adenosine, we generated mice with a targeted disruption of the third coding exon of Cd73 to test the hypothesis that CD73-generated extracellular adenosine functions in an innate protective pathway for hypoxia-induced vascular leakage. Cd73(-/-) mice bred and gained weight normally, and appeared to have an intact immune system. However, vascular leakage was significantly increased in multiple organs, and after subjection to normobaric hypoxia (8% O(2)), Cd73(-/-) mice manifested fulminant vascular leakage, particularly prevalent in the lung. Histological examination of lungs from hypoxic Cd73(-/-) mice revealed perivascular interstitial edema associated with inflammatory infiltrates surrounding larger pulmonary vessels. Vascular leakage secondary to hypoxia was reversed in part by adenosine receptor agonists or reconstitution with soluble 5'-nucleotidase. Together, our studies identify CD73 as a critical mediator of vascular leakage in vivo.

5'-Nucleotidase↗
PubMed1997

Short-term zinc supplementation in women with non-insulin-dependent diabetes mellitus: effects on plasma 5'-nucleotidase activities, insulin-like growth factor I concentrations, and lipoprotein oxidation rates in vitro.

Non-insulin-dependent diabetes mellitus (NIDDM) may cause vulnerability to moderate zinc deficiency. In this study, short-term zinc supplementation (30 mg/d as amino acid chelate for 3 wk) elevated plasma zinc and activities of 5'-nucleotidase, a zinc-dependant enzyme, in 20 postmenopausal women with NIDDM. Placebo, given to 20 other women with NIDDM, had no effects on these indexes nor on any others taken in this study. Although zinc supplementation doubled the mean value for 5'-nucleotidase activity, values were still significantly lower than those of age-matched control subjects. Plasma insulin-like growth factor I concentrations increased with zinc treatment if starting concentrations were < 165 microg/L but were unchanged if they were > 165 microg/L. Lipoprotein oxidation in vitro, which has abnormal lag times and propagation rates for subjects with NIDDM and for moderately zinc-deficient rats, were unchanged by zinc supplementation. Possibly, this lack of effect occurred because the zinc treatment did not normalize zinc status. In conclusion, this study supports the contention that moderate zinc deficiency occurs frequently in subjects with NIDDM.

5'-Nucleotidase↗
PubMed1975

A simple method for preparation of snake venom phosphodiesterase almost free from 5'-nucleotidase.

A simple method, involving NAD+-Sepharose chromatography, was developed for the preparation of snake venom phosphodiesterase (EC 3.1.4.1) almost free from 5'-Nucleotidase (EC 3.1.3.5). Using an NAD+-Sepharose 4B column, phosphodiesterase was eluted in the unadsorbed fraction, whereas 5'nucleotidase was strongly adsorbed. The latter enzyme was desorbed when 0.2 M sodium bicarbonate buffer containing 1mM beta-NADH was used as a solvent. The affinity column could be used at least four times without any decrease of potency, and the method was applicable for the preparation of phosphodiesterase from the venoms of rattlesnake (Crotalus adamanteus) and Japanese mamushi (Agkistrodan halys blomhoffi).

Animals↗
PubMed2000

Cytosolic 5'-nucleotidase hyperactivity in erythrocytes of Lesch-Nyhan syndrome patients.

Lesch-Nyhan syndrome is a metabolic-neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch-Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5'-nucleotidase activity in the erythrocytes of seven patients with Lesch-Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5'-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch-Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.

5'-Nucleotidase↗
PubMed1979

Distribution of erythrocyte nucleotides in pyrimidine 5'-nucleotidase deficiency.

In pyrimidine 5'-nucleotidase deficiency, erythrocytes contain elevated levels of pyrimidine nucleotides. The composition of this nucleotide pool was examined by ion exchange chromatography on Dowex formate columns using a linear ammonium formate elution gradient. In contradistinction to normal erythrocytes, adenine nucleotides accounted for only 32% of the nucleotide pool. The remainder consisted of 50% cytidine and 16% uridine nucleotides. The remaining 2% was not identified. The most abundant compound appeared to be UDP glucose whilst high levels of CTP, CMP and an unidentified cytidine compound less polar than CMP accounted for most of the cytidine nucleotide pool. The possibility that the abnormal nucleotides were due to an elevated reticulocyte count was excluded and it was also shown that erythrocytes from subjects heterozygous for pyrimidine 5'-nucleotidase deficiency did not have detectable levels of the abnormal nucleotides.

Adenosine Triphosphate↗
PubMed2006

Hereditary pyrimidine 5'-nucleotidase deficiency: from genetics to clinical manifestations.

Hereditary pyrimidine 5'-nucleotidase (P5'N) deficiency is the most frequent abnormality of the red cell nucleotide metabolism causing hereditary non-spherocytic haemolytic anaemia. The disorder is usually characterised by mild-to-moderate haemolytic anaemia associated with the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. The precise mechanisms leading to the destruction of P5'N deficient red cells are still unclear. The pyrimidine 5'-nucleotidase type-I (P5'N-1) gene is localised on 7p15-p14 and the cDNA has been cloned and sequenced; 20 mutations have been identified so far in 30 unrelated families, most of them at the homozygous level. Recently, the comparison of recombinant mutants of human P5'N-1 with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and help to correlate genotype to clinical phenotype.

5'-Nucleotidase↗
PubMed1978

Purification and properties of 5'-nucleotidase from lymphocyte plasma membranes.

5'-Nucleotidase is purified from lymphocyte plasma membranes by two affinity chromatographies. The first one, on Lens culinaris lectin-Sepharose 4B yields a fraction of twelve lectin-binding glycoproteins (lectin-receptor fraction). The second one on 5'-AMP-Sepharose 4B leads to pure enzyme. This enzyme is a glycoprotein with a molecular weight of 130 000; it gives a single band in polyacrylamide/dodecylsulfate electrophoresis and displays a very high specific activity (2500-3000 mumol Pih-1mg-1). Some properties of purified 5'-nucleotidase are similar to those of membrane-bound enzyme: substrate specificity, temperature dependence, effects of ions and SH-blocking reagents. Others are completely different for the two systems and these differences result from an interaction between the enzyme molecule and other Lens culinaris lectin binding proteins.

Animals↗
PubMed2004

Mechanistic studies on bovine cytosolic 5'-nucleotidase II, an enzyme belonging to the HAD superfamily.

Cytosolic 5'-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho-enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5'-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.

5'-Nucleotidase↗
PubMed1991

Soluble 5'-nucleotidase activities in rat brain.

5'-Nucleotidase activity was assayed in 105,000-g supernatants from rat brain by following conversion of [3H]AMP into adenosine. The effect of ATP on this process was complex and suggested the presence of at least two soluble 5'-nucleotidase activities: one inhibited by ATP and another activated by ATP. The relative proportions of these activities differed considerably among brain regions. Activity changes induced by hypothyroidism also suggested that these activities may be regulated independently. These findings may have consequences for the regional regulation of adenosine formation in the brain.

5'-Nucleotidase↗