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PubMed1989

The application of affinity chromatography for the separation of "high Km" and "low Km" 5'-nucleotidase and other AMP metabolizing enzymes.

AMP-sepharose 4B has been widely used as a general ligand affinity chromatography for purification of AMP deaminase, 5'-nucleotidase, adenosine kinase and other adenine nucleotide metabolizing enzymes. Since these enzymes generally differ in their kinetic properties related to the values of Km for AMP and analogous compounds, it was assumed that there may be a specific elution pattern of some of the enzymes which would enable sequential elution from the column during a single run. Using 0.5 M NaCl, 10 mM ATP and 5 mM adenosine as eluting agents, it was possible to separate on AMP-sepharose column AMP deaminase "high Km" and "low Km" 5'-nucleotidase and adenosine kinase. Adenylate kinase, adenosine deaminase and nonspecific phosphatase did not bind to the column. Using human placental extract, AMP deaminase, "high Km" and "low Km" 5'-nucleotidase and adenosine kinase were purified 2.8, 2.9, 105 and 1240 fold, respectively. AMP deaminase and "high Km" 5'-nucleotidase were further separated using phosphocellulose column chromatography and the final purification was 227 and 143 fold, respectively. The specific activities of purified enzyme preparations were 9.1, 1.0, 0.4 and 0.5 mumols/min/mg protein of AMP deaminase, "high Km" 5'-nucleotidase and adenosine kinase, respectively. This approach provides a rapid method for initial purification of these enzymes from crude soluble extracts.

5'-Nucleotidase↗

PubMed1980

Histochemical evaluation of lymphocytes in hypogammaglobulinemia. Decreased number of 5'-nucleotidase-positive cells.

With the use of a histochemical technique, 5'-nucleotidase activity was examined in peripheral blood lymphocytes from patients with primary hypogammaglobulinemia. Our current study demonstrates that the decrease in 5'-nucleotidase activity in congenital agammaglobulinemia, previously demonstrated by a radiochemical assay, is associated with a reduction in the number of cells containing 5'-nucleotidase rather than with a decrease of the enzyme activity per cell. Both sheep erythrocyte rosette-forming and nonrosette-forming PBMs have reduced percentages of 5'-nucleotidase-containing cells in subjects with the enzyme deficiency. The reduced percentage of 5'-nucleotidase-containing mononuclear cells in patients with congenital agammaglobulinemia was evident in both monocyte-contaminated and monocyte-depleted cell preparations.

Adult↗

PubMed1983

[Activity of cell-detached 5'-nucleotidase in thymocytes of various densities].

The rat thymocytes submitted to heating at 45 degrees C for 1 hr liberate plasma membrane fragments containing 5'-nucleotidase activity in the supernatant. The thymocytes were separated by ficoll density gradient centrifugation. High activity of 5'-nucleotidase per 10(6) cells was found in the supernatant of low density (1.069) subset of thymocytes. Thymocyte supernatant of rats treated with hydrocortisone demonstrated higher 5'-nucleotidase activity per 10(6) cells than in intact animals. This is due to an increase of the low density population of thymocytes in treated rats since the 5'-nucleotidase activity per 10(6) cells of the supernatant obtained from this density fraction is the same both in treated with hydrocortisone and intact rats. Hydrocortisone seems to induce a selection of the thymocytes with high 5'-nucleotidase activity.

Animals↗

PubMed1994

Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization.

Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.

5'-Nucleotidase↗

PubMed1976

Ecto-5'-nucleotidase of intact cultured C6 rat glioma cells.

The characteristics of 5'-nucleotidase in a clonal line (C6) of rat glioma cells has been examined in detail. The cells liberated 6.80 +/- 0.33 mumol of inorganic phosphate/mg of cell protein/hour, producing nearly equimolar amounts of adenosine and inorganic phosphate from AMP in the extracellular fluid. No 5'-nucleotidase was released by the cells into the medium. Most of the 5'-nucleotidase activity was found to be located in the outer surface of the plasma membrane of C6 cells and rapidly accessible to exogenous AMP, by experiments based upon differential labeling of extracellular and intracellular compartments with 32P and 33P. The ecto-enzyme was active in the absence of divalent cations. However, Mn2+ or Co2+ were somewhat stimulatory. Zn2+ suppressed activity very markedly. The relationship of enzymatic reaction velocity to pH was complex, with an optimum at pH 7.4 for all substrates tested. The ecto-5'-nucleotidase readily hydrolyzed 5'-AMP and 5'-UMP. Other 5'-nucleoside monophosphates, including 5'-deoxy-AMP, were also hydrolyzed, but more slowly; 2'- or 3'-nucleoside monophosphates were not attacked. The ecto-5'-nucleotidase in the intact cell obeyed Michaelis-Menten kinetics. Apparent Km for AMP was 0.22 mM; apparent Km values for other substrates were similar and ranged from 0.16 to 0.18 mM. ADP exerted a very powerful inhibitory effect, behaving as a competitive inhibitor, and 5'-UMP behaved as a strictly competitive substrate for 5'-AMP. ATP and ITP were inhibitory. Of these, ITP served to increase Km for AMP. ATP did likewise, but also greatly lowered Vmax. These findings indicate that the intact cell is capable of rapid hydrolysis of exogenous 5'-AMP, to produce adenosine at the cell surface at a rate which responds directly to extracellular AMP concentration but which can be suppressed by extracellular ADP or ATP.

Binding, Competitive↗

PubMed1994

Electrophoretic fractionation of 5'-nucleotidase.

Human isonucleotidases were separated by electrophoresis on a cellulose acetate membrane. Three 5'-nucleotidase forms, NTP1, NTP2, and NTP3, were resolved with this method and quantified by densitometry. The procedure was not only simple and rapid but also sufficiently precise (between-run CV < 20%) and sensitive (detected nucleotidase fractions of > 0.5 U/L). The effects of various treatments (heat, neuraminidase, glycosidases, proteases, lectins, and detergents) on the electrophoretic pattern of 5'-nucleotidase were studied. NTP1 (mean 12% of total 5'-nucleotidase, SD 5%), NTP2 (mean 30%, SD 8%), and NTP3 (mean 58%, SD 8%) were found in all normal persons studied. The increase in total 5'-nucleotidase in patients with hepatobiliary disease was mainly due to the NTP1 isoform.

5'-Nucleotidase↗

PubMed1995

Crucial role of ecto-5'-nucleotidase in differentiation and survival of developing neural cells.

ATP, an important signalling substance in the central nervous system, is hydrolysed to adenosine via a surface-located enzyme cascade. The final hydrolysis step from AMP to adenosine is catalysed by 5'-nucleotidase, a GPI-anchored surface protein. 5'-Nucleotidase is transiently expressed on developing neurones. An antisense oligonucleotide that suppresses the synthesis of 5'-nucleotidase inhibits NGF-induced neurite formation and survival in PC12 cells and cultures of cerebellar granule cells. The inhibitory effect of the antisense oligonucleotide can be completely or partially relieved by addition of soluble 5'-nucleotidase or of nucleosides to the medium. Our results suggest that 5'-nucleotidase is essential for the differentiation and survival of neural cells and represents an important and critical step in the hydrolysis cascade of extracellular nucleotides.

5'-Nucleotidase↗

PubMed1987

Single-strand-specific nuclease of pea seeds: glycoprotein nature and associated nucleotidase activity.

A single-strand-specific nuclease from germinating pea seeds has been purified to homogeneity. The purification procedure includes affinity chromatography on concanavalin A-Sepharose and gel filtration. The nuclease exhibits its activity at neutral pH and does not have an absolute requirement for a divalent cation. The purified nuclease also possesses a 3'-nucleotidase activity and is a glycoprotein containing about 20% carbohydrate. On native polyacrylamide gels the nuclease activity comigrates with the nucleotidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits in the native enzyme. The nuclease and nucleotidase activities show differential rates of thermal inactivation, the latter following simple first order kinetics and the former exhibiting a more complex reaction. The nucleotidase was also found to be stimulated by DNA, the increase being greater with native DNA than with denatured DNA. These properties are possibly accounted for by the dimeric structure of the enzyme where the nucleotidase catalytic site resides in one subunit while the nuclease site is formed by interaction of both subunits. The enzyme also hydrolyzes double-stranded alkylated DNA and depurinated DNA at a higher rate than native DNA. Experimental evidence suggests that depurinated DNA is hydrolyzed in the region of apurinic sites.

Chromatography, Affinity↗

PubMed2004

ATP- and adenosine-mediated signaling in the central nervous system: adenosine receptor activation by ATP through rapid and localized generation of adenosine by ecto-nucleotidases.

Extracellular ATP is now recognized as a neurotransmitter or neuromodilator in the nervous system, producing diverse physiological effects by activating multiple P2 receptors. Although P2-receptor signaling is terminated by hydrolysis of ATP by the ecto-nucleotidase cascade, such a metabolic step leads to adenosine generation, thereby initiating adenosine (P1)-receptor activation. Because most cells and tissues co-express P1 and P2 receptors, ecto-nucleotidase on target tissues, especially enzymes catalyzing adenosine formation, are determinants of the cellular response to ATP. Ecto-5'-nucleotidase (E-5'-NT) has been considered to play a principal role in conversion of AMP to adenosine. In addition to E-5'-NT, we have recently demonstrated that ecto-alkaline phosphatase is also involved in ATP-induced P1-receptor activation through a rapid and localized adenosine production on the membrane surface. In this minireview, we describe the pharmacological profile of ecto-nucleotidase-dependent P1-receptor activation by ATP and molecular bases of preferential delivery of metabolically generated adenosine to P1 receptors. Several lines of evidence suggest that the close association between ecto-nucleotidases and P1 receptors may constitute a functional receptor for extracellular ATP, and some physiological responses to ATP would occur through this mechanism.

Adenosine↗

PubMed1988

Qualitative and quantitative differences between Burkitt lymphoma and lymphoblastoid cell lines in the expression of membrane ecto-nucleotidases and in the response to agonists of the A2-type adenosine receptor.

The activity of ecto-nucleotidases has been determined on intact cells from Burkitt lymphoma (BL) and lymphoblastoid cell lines established in vitro (LCLs). BL cells never express ecto-5'-nucleotidase (5'-N) and exhibit only low levels of ecto-ATPase activity, whereas LCL-cells usually express both enzymes to varying degrees. There is a certain correlation (r = 0.75) between 5'-N and ecto-ATPase. When cAMP formation in response to agonists of the A2-type (stimulating) adenosine receptor is measured in the same cell lines there is no correlation with the expression of ecto-nucleotidases. Within pairs of BL and LCL cell lines derived from the same donor, an inverse relationship between ecto-nucleotidases and the response to the adenosine receptor agonist N-ethyl-carboxamido-adenosine (NECA) is observed. BL cells show a good response to NECA, whereas this is low or absent in LCL cells. Treatment of cells which exhibit both 5'-N and the adenosine receptor with specific polyclonal or monoclonal antibodies against the enzyme does not impair the function of the receptor. Antisera against peptides of the membrane antigen BNLF I-MA, coded by the EBV-genome, do not co-precipitate 5'-N out of detergent extracts of LCL-cells. In both cases 5'-N cannot be closely associated with other membrane components. The differences between BL and LCL cells in ectonucleotidases and the adenosine receptor appear to be fairly stable phenotypical markers, independent of other parameters and suitable for use in distinguishing these cells in all populations.

5'-Nucleotidase↗

PubMed1987

Diurnal variation in 5'-nucleotidase activity in rat liver. A quantitative histochemical study.

The diurnal variation of 5'-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5'-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5'-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5'-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.

5'-Nucleotidase↗

PubMed1983

5'-Nucleotidase activity in human astrocytomas.

Human astrocytomas contain 5'-nucleotidase as shown using histochemical, cytochemical and biochemical techniques. 5'-Nucleotidase activity was revealed by electron microscopy to be associated with the outer surface of the plasma membrane of astrocytoma cells. The results confirm and extend previous reports of the localisation of 5'-nucleotidase in normal astrocytes in situ. Biochemical assay of 5'-nucleotidase activity in 35 tumours showed it to be clearly higher in malignant (anaplastic astrocytomas and glioblastomas) than in benign astrocytomas.

5'-Nucleotidase↗

PubMed1983

Ecto-5'-nucleotidase regeneration after chemical modification of the plasma membrane.

Treatment of the C6 glioblastoma cell with trinitrobenzenesulfonic acid (TNBS) resulted in the selective inactivation of ecto-5'-nucleotidase under conditions which maintained cell viability. Cells respond to ecto-enzyme inactivation by replacing 80% of lost activity within 24 hrs. A lag time of 4-6 hrs before ecto-5'-nucleotidase replacement began and its complete blockage by cycloheximide indicated that the source of replaced enzyme was de novo synthesis and not an intracellular pool. Release of 5'-nucleotidase activity into culture medium in the form of membraneous vesicles slowed during the active recovery period and then steadily increased with time as the plasma membrane enzyme level approached normal. TNBS did not exert a direct inhibitory action upon the exfoliative process as release of vesicular GM1 and protein were little affected. Decrease in exfoliated 5'-nucleotidase activity may be due to a selective conservation of the enzyme in the exfoliative process.

5'-Nucleotidase↗

PubMed1981

5'-Nucleotidase activity in lymphocytes.

Some characteristics of lymphocyte 5'-nucleotidase are reviewed. The optimal conditions for the cytochemical localization of 5'-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that cytochemistry can be performed under conditions that do not lead to loss of AMPase activity. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. The splenic cell population included a distinct population of lymphocytes with readily demonstrable AMPase activity and another population with no cytochemically demonstrable AMPase activity. The number of positive cells varied, but was usually between 20 and 30% when cells were fixed in glutaraldehyde. Lymphocytes purified from thymus were always negative for cytochemically demonstrable AMPase activity. Biochemically, it was shown that the AMPase activity of spleen lymphocytes is more than six times higher than that of thymus lymphocytes. 5'-Nucleotidase activity in normal and abnormal lymphocytes is discussed in the light of the latest findings. The possible function of 5'-nucleotidase in lymphocytes is discussed.

5'-Nucleotidase↗

PubMed1988

Pitfalls in ecto-5'-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages.

We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5'-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killed Escherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5'-nucleotidase onto the adhering particle was fact or artifact, we localized 5'-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5'-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.

5'-Nucleotidase↗

PubMed1986

5'-nucleotidase activity of murine T-cell subpopulations separated according to their Lyt2 and L3T4 phenotypes.

Murine T lymphocytes were separated by "panning" into four subpopulations, according to their Lyt2 and L3T4 phenotypes: Lyt2+L3T4+, Lyt2-L3T4-, Lyt2+L3T4-, and Lyt2-L3T4+. The activity of ecto-5'-nucleotidase in each subpopulation was measured. 5'-Nucleotidase activity was undetectable in Lyt2+L3T4+ cortical cells but was expressed in medullary Lyt2-L3T4+ and Lyt2+L3T4- T lymphocytes. The small cortical subpopulation of thymocyte precursors with the Lyt2-L3T4- phenotype expressed levels of 5'-nucleotidase comparable to the levels of medullary, mature lymphocytes. These results suggest that the use of ecto-5'-nucleotidase as a marker of the degree of T-cell maturation is questionable.

5'-Nucleotidase↗

PubMed1993

Characteristics of membrane-bound 5'-nucleotidase on human prostasomes.

Examination of prostasomes, isolated from human seminal plasma, showed that there was very little remaining paranitrophenylphosphatase activity when assayed in the presence of 10 mmol/l of tartrate and 2 mmol/l of levamisole. Under these conditions it was possible to study the prostasome membrane-bound 5'-nucleotidase activity, which was unaffected by these two inhibitors. The activity was considered to be located at the external surface of the prostasome membrane and a 50-60% increase in activity was obtained by the addition of 0.05% Triton X-100. The prostasome membrane-linked 5'-nucleotidase readily hydrolysed 5'-AMP. Two other 5'-nucleoside monophosphates, 5'-IMP and 5'-GMP, were also hydrolysed, but more slowly; 2'- or 3'-AMP were practically not attacked. The prostasome membrane-linked 5'-nucleotidase obeyed Michaelis-Menten kinetics. Apparent Km for 5'-AMP was 11.2 +/- 2.1 mumol/l and Vmax 64.7 +/- 11.4 nmol/mg protein/min. These figures were somewhat changed in presence of 0.05% Triton X-100, the Km value being reduced by 30% and the Vmax value increased by 60%. Adenosine 5' (alpha, beta methylene) diphosphate (100 mumol/l), Ni2+ (10 mmol/l) and concanavalin A (20 micrograms/ml) were all potent inhibitors of the prostasome membrane-linked 5'-nucleotidase.

5'-Nucleotidase↗

PubMed1983

Localisation of 5'-nucleotidase in a rat liver cell line using a monoclonal antibody and indirect immunofluorescent labelling.

A new method of labelling anti-IgG with rhodamine while protecting the IgG binding site is described. Indirect immunofluorescence of the ectoenzyme 5'-nucleotidase using a monoclonal antibody shows that it has a punctate distribution over the whole cell surface of a cultured rat liver cell line. The density of fluorescent spots representing the surface localisation of 5'-nucleotidase and of clathrin-coated pits was analysed by a computerised counting technique. The cell surface localisation of 5'-nucleotidase differs from clathrin-coated pits and microvilli. 5'-Nucleotidase within the cell is not uniquely associated with any clearly defined organelle.

5'-Nucleotidase↗