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PubMed1977

Filter paper disk techniques for assay of nucleotidase.

A DE filter disk technique for assaying the activity of nucleotidase is described. This method is based on the observation that nucleotides bind to the filters at 5 mM Tris-HCl (pH 7.8) while nucleosides do not. As parameter for the nucleotidase activity the decrease of bound nucleotides is determined. In parallel experiments the amount of the product (nucleoside) formed can be measured by DEAE Sephadex column chromatography. The filter disk technique can be applied for the determination of vmax and Km of a nucleotidase by using different ribonucleosidase monophosphate substrates.

Adenosine Monophosphate↗

PubMed1978

Cytochemical investigations on the localization of 5'-nucleotidase in the rat hippocampus with special reference to synaptic regions.

The fine structural localization of the 5'-nucleotidase was investigated in the CA3 region of the rat hippocampus. The attention was focussed on the occurrence of the enzyme in the synaptic region. The 5'-nucleotidase activity was demonstrated at the surface membranes of axons and dendrites. Prominent portions of enzyme activity were detectable in the nuclei and the nuclear envelope, whereas the cytoplasmic organelles were nearly devoid of reaction product. In synapses five types of 5'-nucleotidase localization were revealed. A participation of the enzyme in the process of neurotransmission is discussed.

Animals↗

PubMed1978

Electron microscopic localization of 5'-nucleotidase in the stratum intermedium and ameloblasts.

5'-nucleotidase was demonstrated at the fine structural level in the stratum intermedium and ameloblasts of the first mandibular molars of CD-1 mice. The enzyme was localized with the Wachstein & Meisel (1957) method along the plasma membranes of the cells of the stratum intermedium and ameloblasts. While 5'- nucleotidase was present throughout the stratum intermedium, only the proximal region of the plasma membranes of ameloblasts was demonstrably active for this enzyme. 5'-Nucleotidase has been implicated in transport of metabolites across cell membranes, and its localization in the present study supports this implication as well as the transport functions of the stratum intermedium and the stratum intermedium--ameloblastic interface.

Ameloblasts↗

PubMed1999

Evidence that cytosolic and ecto 5'-nucleotidases contribute equally to increased interstitial adenosine concentration during porcine myocardial ischemia.

The purpose of this study was to determine the roles of cytosolic and ecto 5'-nucleotidase in myocardial ischemia-induced increases in interstitial fluid (ISF) adenosine. Pentobarbital anesthetized, open chest pigs were instrumented with two microdialysis fibers in the distally perfused bed of the left anterior descending (LAD) coronary artery to estimate ISF metabolites. Fibers in control hearts were perfused with standard Krebs buffer. In two additional groups, after collecting one dialysate sample with normal Krebs, fibers were perfused with buffer supplemented with either L-homocysteine thiolactone (5 mM) or the ecto 5'-nucleotidase inhibitor alpha, beta-methylene adenosine 5'-diphosphate (AOPCP, 5 mM). Hearts were then submitted to 60 minutes LAD occlusion and two hours reperfusion. Dialysate nucleosides and AMP were measured by high performance liquid chromatography. The local delivery of homocysteine did not alter preischemic dialysate adenosine concentration (0.30 +/- 0.04 microM) compared to pre-homocysteine infusion (0.39 +/- 0.04 microM) or control hearts (0.36 +/- 0.04 microM), but AOPCP significantly decreased preischemic dialysate adenosine levels (from 0.36 +/- 0.02 to 0.14 +/- 0.03 microM). During LAD occlusion both homocysteine and AOPCP reduced dialysate levels by approximately 50%. At 30 minutes ischemia dialysate adenosine concentrations were 19.47 +/- 2.72, 11.41 +/- 2.44, and 7.93 +/- 1.01 microM in control, homocysteine, and AOPCP hearts, respectively. AOPCP significantly increased dialysate AMP levels; at 60 minutes ischemia AMP levels were 6.22 +/- 2.97 microM in control hearts and 38.60 +/- 5.69 microM in AOPCP treated hearts. These results suggest that both cytosolic and ecto 5'-nucleotidase contribute to ischemia-induced increases in ISF adenosine in porcine myocardium.

5'-Nucleotidase↗

PubMed2006

Apyrase and 5'-nucleotidase activities in synaptosomes from the cerebral cortex of rats experimentally demyelinated with ethidium bromide and treated with interferon-beta.

Apyrase and 5'-nucleotidase activities were analyzed in an ethidium bromide (EB) demyelinating model associated with interferon-beta (IFN-beta). The animals were divided in groups: I, control (saline); II, saline and IFN-beta; III, EB and IV, EB and IFN-beta. After 7, 15 and 30 days the animals (n = 5) were sacrificed and the cerebral cortex was removed for synaptosome preparation and enzymatic assays. Apyrase activity using ATP as substrate increased in groups II, III and IV (P < 0.001) after 7 days and in groups III and IV (P < 0.001) after 15 days. Using ADP as substrate, an activation of this enzyme was observed in group III (P < 0.05) after seven and 15 days. The 5'-nucleotidase activity increased in group III (P < 0.05) after 7 days and in groups II, III and IV (P < 0.001) after 15 days. After 30 days treatment, no significant alteration was observed in enzyme activities. Results showed that apyrase and 5'-nucleotidase activities are altered in demyelination events and that IFN-beta was able to regulate the adenine nucleotide hydrolysis.

5'-Nucleotidase↗

PubMed1977

Contrast manifestation of alkaline phosphatase and 5'-nucleotidase in plasma membranes isolated from rat liver and ascites hepatoma.

1. Plasma membranes were isolated from ascites hepatoma AH-130 and rat livers with or without partial hepatectomy or bile duct ligation. Reciprocal manifestations of two marker enzymes for plasma membranes were observed in these membrane preparations; alkaline phosphatase activity was found much higher in the hepatoma membrane than in any preparations of the liver membranes, whereas 5'-nucleotidase activity was much lower in the former than in the latter. 2. Effects of lectins and anti-plasma membrane antiserum on these two marker enzymes were examined. The results showed that about 50% of apparent activity of 5'-nucleotidase found in the hepatoma membrane was exhibited by alkaline phosphatase. 3. Localizations of alkaline phosphatase and 5'-nucleotidase in polyacrylamide gels after electrophoresis were demonstrated using 5'-AMP and 5-Br, 4-Cl-indoxylphosphate as substrate. There was a difference in electrophoretic mobility between the alkaline phosphatase of the hepatoma and that of the liver.

Adenosine Monophosphate↗

PubMed1978

The Arrhenius plot behaviour of rat liver 5'-nucleotidase in different lipid environments.

The plasma membrane enzyme 5'-nucleotidase (EC 3.1.3.5) was prepared from rat liver as a complex with sphingomyelin or in detergent-solubilized forms. Each preparation exhibited a Km and Arrhenius break temperature indistinguishable from that of 5'-nucleotidase in plasma membranes. Measurement of fatty acid profiles, cholesterol and phospholipid content however showed a very wide variation between these preparations. We conclude that the biphasic nature of the Arrhenius plot of 5'-nucleotidase may be a property of the enzyme rather than its lipid environment.

Cell Membrane↗

PubMed1994

Mutational analysis of the COOH-terminal hydrophobic domain of bovine liver 5'-nucleotidase as a signal for glycosylphosphatidylinositol (GPI) anchor attachment.

In order to address the minimum domain of the COOH-terminal hydrophobic region responsible for GPI modification of bovine liver 5'-nucleotidase, we constructed a series of the deletion mutants of the COOH-terminus and expressed them in COS cells. Cells transfected by the deletion mutant of 6 amino acids (-IIILYQ) from the hydrophobic domain (-FSLIFLSVLAVIII-LYQ) did not show any elevation of cell surface-associated 5'-nucleotidase activity, whereas the 2 (-YQ) or 4 (-ILYQ) amino acid deletion mutant retained the bovine liver-derived activity on the cell surface as a GPI-anchored protein. Loss of half the hydrophobic domain (6 or 8 amino acids) resulted in accumulation of the activity in the cell. On the other hand, deletion of the whole hydrophobic domain (17 amino acids) or the entire cleaved-off domain (25 amino acids) made the product secreted into the medium. In conclusion, the hydrophobicity of 13 amino acids in length was enough for the GPI modification of the bovine liver 5'-nucleotidase.

5'-Nucleotidase↗

PubMed1976

Cytosol 5'-nucleotidase from chicken liver. Purification and some properties.

1. 5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from the cytosol of chicken liver has been purified 1860-fold with an overall yield of 20% by a combination of precipitation at pH 5.3, (NH4)2SO4 fractionation, calcium phosphate gel adsorption, phosphocellulose chromatography and gel filtration with Sephadex G-200. The enzyme has been shown to be highly purified, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This is the first time it has been possible to obtain a purified 5'-nucleotidase from the cytosol of animal tissue. 2. An S20, W of 9.7 S for 5'-nucleotidase was obtained by the use of sucrose density gradient centrifugation and a Stokes radius of 5.1 nm was estimated by gel filtration techniques. From these values and the assumed partial specific volume of 0.725 cm3/g, the molecular weight of the enzyme was calculated to be 205 000. One major band, corresponding to a molecular weight of 51 000, was detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicating that the native enzyme was composed of four identical subunits. 3. Some properties of the purified enzyme, including pH optimum Mg2+ dependency and substrate specificity, resembled closely those of the partially purified enzyme from chicken liver acetone powder as reported by Itoh, R., Mitsui, A. and Tsushima, K. (1967) Biochim. Biophys. Acta 146, 151-159.

Adenosine Monophosphate↗

PubMed1979

Purification and properties of 2'-nucleotidase from mammalian brain.

A nucleotidase specific for 2'-nucleotides was localized in both the soluble and the synaptosomal fractions of rat brain. The enzyme was partially purified from the soluble fraction of bovine brain. The s20,w was 4.9 S with an estimated molecular weight of about 70 000. The optimum pH was 8.0 and Km value for 2'-AMP was 5.5 . 10(-4) M. The substrate and inhibitor specificities of the enzyme were examined. The nucleotidase has an absolute requirement for Mg2+ but neither Fe3+ nor Ca2+ acted as replacement ions. In fact, Mn2+ and Ca2+ inhibited the Mg2+-dependent 2'-nucleotidase.

Adenosine Monophosphate↗

PubMed1991

Cytochemistry of 5'-nucleotidase in the superior cervical ganglion of cat and guinea pig.

The localization of 5'-nucleotidase, an adenosine-producing ectoenzyme, was studied by a cytochemical method in the superior cervical ganglion of the adult cat and guinea pig. The following subcellular sites of enzymatic activity were detected: (1) the surface of Schwann and satellite cells including the extracellular space between these cells and neuronal profiles; (2) the plasmalemma and pinocytotic vesicles of capillary endothelial cells; and (3) the synaptic clefts between cholinergic preganglionic axon terminals and sympathetic neurons. The simultaneous presence of 5'-nucleotidase at both glial elements and synapses within the adult peripheral nervous system (PNS) constitutes a novel distribution pattern for this enzyme which does not apply to the mature central nervous system (CNS), but which is rather typical for the developing CNS. These distributions of 5'-nucleotidase activity may reflect specific cellular requirements for nucleosides involved in parenchymal metabolism, in vascular transport processes and, possibly, in synaptic plasticity.

5'-Nucleotidase↗

PubMed1977

Electrophoretic characterization of pyrimidine 5'-nucleotidase of human erythrocytes and its distinction from acid phosphatase.

Electrophoresis of red cell pyrimidine 5'-nucleotidase was carried out on cellulose acetate strips. One major band of activity was found in preparations from human erythrocytes. This enzyme showed a specificity for the pyrimidine nucleotides UMP and CMP. No activity was detected with AMP. Pyrimidine 5'-nucleotidase activity could be separated from that of acid phosphatase with the use of alpha-glycerophosphate. This method may be useful in the study of pyrimidine 5'-nucleotidase deficiency in red cells.

Acid Phosphatase↗

PubMed1982

Age-related changes in 5'-nucleotidase and alkaline phosphatase activities in mouse thymocytes.

Enzymatic activities of thymocytes isolated from Swiss albino mice were studied at various ages from immediately post weaning until 100 weeks of age, approaching the life expectancy of these animals. Between 5 and 10 weeks of age, the activities of lactate dehydrogenase and alkaline phosphatase decreased to a level that was maintained throughout the remainder of the aging profile. Neutral beta-glycerophosphatase (pH 7.5) activity in a thymus membrane preparation was similar in all age groups. The activity of membrane-bound 5'-nucleotidase, that is, AMP-hydrolyzing activity inhibited by 100 microM alpha, beta-methyleneadenosine 5'-diphosphate, progressively increased as a function of age, indicating thymocyte population changes occurring very late in life. In thymocytes of the oldest mice examined (100 weeks of age), 5'-nucleotidase specific activity was approximately ten-fold greater than the activity found in 5-week-old mice. Thus, membrane-bound 5'-nucleotidase activity in thymocytes increased markedly as a function of age in Swiss albino mice; yet several other enzymatic activities, including alkaline phosphatase, remained relatively unchanged in mature mice.

Aging↗

PubMed1992

A novel non-radioactive method for detection of nucleoside analog phosphorylation by 5'-nucleotidase.

Cytosolic 5'-nucleotidase has been implicated in the phosphorylation of certain nucleosides of therapeutic interest. In vitro, IMP and GMP serve as the optimal phosphate donors for this nucleoside phosphotransferase reaction. Existing assays for nucleoside phosphorylation effected by 5'-nucleotidase require a radiolabeled nucleoside as the phosphate acceptor and separation of the substrate-nucleoside from product-nucleotide has been accomplished either by a filter binding method or HPLC. However, detection of the phosphorylation of unlabeled nucleoside by HPLC is difficult since the ultraviolet absorbance of the phosphate donor, IMP, frequently obscures the absorbance of newly formed nucleotide. The use of ribavirin 5'-phosphate (RMP, 1,2,4-triazole-3-carboxamide riboside 5-monophosphate) as the phosphate donor obviates this difficulty since this triazole heterocycle does not significantly absorb at the wavelengths used to detect most nucleoside analogs. Using this procedure, a 5'-nucleotidase activity from the 100,000 x g supernatant fraction of human T-lymphoblasts deficient in adenosine kinase, hypoxanthine-guanine phosphoribosyltransferase, and deoxycytidine kinase, was characterized with regard to structure-activity relationships for certain inosine and guanosine analogs.

5'-Nucleotidase↗

PubMed1996

Synergistic action of ADP and 2,3-bisphosphoglycerate on the modulation of cytosolic 5'-nucleotidase.

Cytosolic 5'-nucleotidase, acting preferentially on IMP, GMP and their deoxyderivatives, can also behave as a phosphotransferase, operating a transfer of phosphate from a nucleoside monophosphate donor to a nucleoside acceptor which, besides a natural nucleoside, can be also an analog. The enzyme activity is stimulated by ADP, ATP and 2,3-bisphosphoglycerate (BPG). The concentration of effector required to attain half maximal activation (A0.5) for the bisphosphorylated compound is in the millimolar range, so that BPG seems to act as a physiological activator of 5'-nucleotidase only in erythrocytes. However, the combination of BPG and ADP brings about a significant increase of their respective affinity for the enzyme, lowering their A0.5 values approx. 4-times. The observation that BPG favors the phosphotransferase more than the hydrolase activity of 5'-nucleotidase stands for a key role of this metabolite in the regulation of the processes of activation of purine pro-drugs, in which this enzyme is involved.

2,3-Diphosphoglycerate↗

PubMedDate not supplied

5'-Nucleotidase activity increases in aging rat brain.

The enzyme 5'-nucleotidase is present in glial and neuronal membranes, and catalyzes the formation of adenosine, which in turn can act as a neuromodulator or neurotransmitter. The present study found marked increases in histochemically demonstrated 5'-nucleotidase activity in most regions of rat brain from young adulthood (3-4 months) to middle age (12-18 months), with smaller or no changes between middle and old age (24-32 months). The aging adult cerebellum showed alterations in the histochemical pattern, with declines in the molecular layer and increases in the Purkinje layer. Both myelin and synaptic plasma membrane fractions from forebrain showed increases in enzyme activity. Assays of various other body tissues suggested that the increases are fairly specific to brain, and thus apparently do not represent a ubiquitous cellular mechanism of aging. Changes in brain 5'-nucleotidase activity during aging probably reflect the increasing number and size of glial cells, and perhaps also affect synaptic transmission through regulation of adenosine.

5'-Nucleotidase↗

PubMed1996

Biochemistry, localization and functional roles of ecto-nucleotidases in the nervous system.

Nucleotides such as ATP, ADP, UTP or the diadenosine polyphosphates and possibly even NAD+ are extracellular signaling substances in the brain and in other tissues. Enzymes located on the cell surface catalyze the hydrolysis of these compounds and thus limit their spatio-temporal activity. As a final hydrolysis product they generate the nucleoside and phosphate. The paper discusses the biochemical properties, cellular localization and functional properties of surface-located enzymes that hydrolyse nucleotides released from nervous tissue. This is preceded by a brief discussion of nucleotide receptors, cellular storage and mechanisms of nucleotide release. In nervous tissue nucleoside 5'-triphosphates are hydrolysed by ecto-ATP-diphosphohydrolase and possibly in addition also by ecto-nucleoside triphosphatase and ecto-nucleoside diphosphatase. The molecular identity of the ATP-diphosphohydrolase has now been revealed. The hydrolysis of nucleoside 5'-monophosphates is catalysed by 5'-nucleotidase whose biochemical properties and molecular structure have been studied in detail. Little is known about the molecular properties of the diadenosine polyphosphatases. Surface located enzymes for the extracellular hydrolysis of NAD+ and also ecto-protein kinases are discussed briefly. The cellular localization of the ecto-nucleotidases is only partly defined. Whereas in adult mammalian brain activity for hydrolysis of ATP and ADP may be associated with nerve cells or glial cells 5'-nucleotidase appears to have a preferential glial allocation in the adult mammal. The extracellular hydrolysis of the nucleotides is of functional importance not only during synaptic transmission where it functions in signal elimination. It plays a crucial role also for the survival and differentiation of neural cells in vitro and presumably during neuronal development in vivo.

5'-Nucleotidase↗

PubMed1993

Hippocampal localization of 5'-nucleotidase as revealed by immunocytochemistry.

The distribution of binding sites for an antibody against ecto-5'-nucleotidase was investigated in the mouse hippocampus by light microscopical immunocytochemistry. The antibody selectively labels a band corresponding to the innervation area of mossy fibre terminals within area CA3. Area CA1 as well as the dendate gyrus are negative. In area CA3 only the proximal but not the distal parts of the apical dendrites of pyramidal cells are labelled. Labelling is in the form of large dots around dendrites of pyramidal cells suggesting that mossy fibre terminals are immunopositive. In contrast, an antibody against the ubiquitous synaptic vesicle protein SV2 labels the large mossy fibre terminals as well as fine and punctate structures in the dendritic and somatic regions throughout the hippocampus. Labelled astrocytes can be found in the entire hippocampus and are frequent in the stratum radiatum and stratum oriens of the CA1 region. Immunopositive astrocytic processes can be found in association with capillary walls. Our results suggest that ecto-5'-nucleotidase may play a crucial role in the hydrolysis of AMP to adenosine at the mossy fibre synapses. Thus, at these synapses, 5'-nucleotidases could function both in completing the extracellular hydrolysis of synaptically released ATP as well as in the extracellular formation of adenosine.

5'-Nucleotidase↗