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PubMed1980

Red cell pyrimidine 5'-nucleotidase and glutathione in myeloproliferative and lymphoproliferative disorders.

Red cell reduced glutatione (GSH) and pyrimidine 5'-nucleotidase (Pry 5'-NT) were measured in a variety of myeloproliferative and lymphoproliferative disorders. Raised levels of GSH were found in chronic lymphocytic leukaemia, Hodgkin's disease, non-Hodgkin's lymphoma, Waldenstrom's macroglobulinaemia and myeloma. Decreased activity of Pyr 5'-NT was found in acute myeloblastic leukaemia, acute lymphoblastic leukaemia, chronic granulocytic leukaemia, chronic lymphocytic leukaemia, Hodgkin's disease and non-Hodgkin's lymphoma. There was no correlation between the raised GSH levels and decrease Pry 5'-NT levels.

Adult↗
PubMed1989

A spectrophotometric method for the assay of pyrimidine 5'-nucleotidase in human erythrocytes.

A new spectrophotometric assay for human erythrocyte pyrimidine 5'-nucleotidase using a continuous spectrophotometric method is described. The activity is detected by measuring uracil, which is produced by a coupled assay including cytidine deaminase and uridine nucleosidase. The results obtained with the continuous spectrophotometric method have been confirmed by high pressure liquid chromatography (HPLC) analysis. This procedure, not based upon inorganic phosphate formation and avoiding the use of radioactive material, offers significant advantages with respect to commonly adopted assays.

5'-Nucleotidase↗
PubMed2004

Molecular basis of Japanese variants of pyrimidine 5'-nucleotidase deficiency.

The type-I isoform of pyrimidine 5'-nucleotidase (P5N-I) has an important role in the catabolism of pyrimidine mononucleotides during erythroid maturation. Two alternatively spliced forms of P5N-I mRNA have been identified, and we found another alternatively spliced form in reticulocytes, which included an additional 87-bp sequence. The sequence is located 6.2-kb downstream of the exon 2 and 2.7-kb upstream of the exon 3 sequence; consequently, the P5N-I gene encodes 11 exons, which span approximately 48 kb. We identified five novel mutations in nine families with P5N-I deficiency: two missense mutations (425C, 721C), one splice mutation (339C), one 1-bp insertion (251-insA-252) and one 9-bp deletion (del 192-200). All patients were homozygous for each mutation. The mutant P5N-I with 721C (G241R) had lower affinity for cytidine monophosphate, suggesting that Gly241 is important for substrate binding. Haplotype analysis showed that 721C, which had been identified in five unrelated families, was a founder mutation. The mutant P5N was then expressed in Cos-7. The degradation of P5N with 425C (L142P) was significantly faster than a wild-type control, and proteasome inhibitors restored the stability of L142P. These data suggest that L142P increases susceptibility to the degradation by the ubiquitin-proteasome pathway.

5'-Nucleotidase↗
PubMed1992

Effects of immunomodulators on ecto-5'-nucleotidase activity on blood mononuclear cells in vitro.

In this study the effects of immunomodulators on the ecto-5'-nucleotidase (ecto-5'-NT) activity on blood mononuclear cells (BMC) were examined in vitro. Interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) decreased the level of ecto-5'-NT activity on BMC whereas prostaglandin E2 (PGE2) increased the ecto-5'-NT level. All three immunomodulators influenced the ecto-5'-NT activity of isolated monocytes whereas only IL-4 and PGE2 had an effect on the enzyme level on isolated lymphocytes. The effect was dependent upon protein synthesis. The effect was dose dependent: IL-4 was effective at concentrations down to 0.5 U/ml, IFN-gamma down to 40 U/ml and PGE2 at nanomolar concentrations. These data indicate that immunomodulators may also take part in the regulation of ecto-5'-NT activity on BMC in vivo. BMC from 7 patients with different immunodeficiency syndromes showed decreased ecto-5'-NT activity on freshly isolated cells. However, following culture ecto-5'-NT activity was increased above the level found on freshly isolated BMC from healthy persons. On BMC from 3 patients with hypogammaglobulinaemia, the effect of IL-4 on the level of ecto-5'-NT activity was identical to that found on BMC from healthy donors, whereas PGE2 increased ecto-5'-NT activity on BMC from only 1 of the 3 patients investigated. The decreased ecto-5'-NT activity of BMC from patients with immunodeficiency may thus be due to a defective regulation of ecto-5'-NT activity in vivo.

5'-Nucleotidase↗
PubMed1990

Production and characterization of monoclonal antibodies to the glycosyl phosphatidylinositol-anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (CD73).

A panel of monoclonal antibodies to the 69 kDa glycosyl phosphatidylinositol anchored lymphocyte differentiation antigen ecto-5'-nucleotidase (ecto-5'-NT, CD73) was produced using highly purified human placental 5'-NT as immunogen. Antibodies 1E9.28.1 and 7G2.2.11 inhibit soluble placental 5'-NT activity and recognize lymphocyte CD73 in indirect immunofluorescence and immunoprecipitation assays. In addition, 1E9.28.1 induces vigorous T cell proliferation in the presence of submitogenic doses of phorbol myristate and F(ab')2 goat anti-mouse Ig. Both antibodies can be used to purify the three major forms of placental 5'-NT by affinity chromatography. By two-color immunofluorescence, CD73 was found to be expressed on 19 +/- 5% of CD3+, 11 +/- 4% of CD4+, 51 +/- 14% of CD8+, 25 +/- 8% of CD28+, 15 +/- 5% of CD29+, 27 +/- 7% of CD45RA+, and 70 +/- 6% of CD19+ lymphocytes. Within T cells, CD73 expression is restricted to the CD28+ subset. Thus, CD73 is found on subsets of both T and B lymphocytes, with the highest expression on B cells and CD8+ T cells. In sections of hyperplastic tonsil, CD73 expression is restricted to the small lymphocytes of the follicular mantle zone, a small subset of extrafollicular lymphocytes situated within the epithelium of the tonsillar crypt, and to follicular dendritic cells within the lower part of the "light-zone." CD73 is also detected on subsets of endothelial cells of capillaries and venules and the basal layer of non-keratinizing squamous epithelium and transitional cell type mucosa of many tissues. Given the tissue distribution of CD73, along with its glycosyl phosphatidylinositol membrane anchoring and the observation that some CD73 antibodies are mitogenic, we propose that this interesting antigen may play a role in cell activation, lymphocyte homing, and/or cell adhesion.

5'-Nucleotidase↗
PubMed1995

Evidence that [3H]-alpha,beta-methylene ATP may label an endothelial-derived cell line 5'-nucleotidase with high affinity.

1. In membranes prepared from a permanent cell line of endothelial origin (WEC cells), [3H]-alpha, beta-methylene ATP ([3H]-alpha, beta-meATP) labelled high (pKd = 9.5; Bmax = 3.75 pmol mg-1 protein) and low (pKd = 7.2; Bmax = 23.3 pmol mg-1 protein) affinity binding sites. The high affinity [3H]-alpha, beta-meATP binding sites in the WEC cell membranes could be selectively labelled with a low concentration of the radioligand (1 nM). In competition studies performed at a radioligand concentration of 1 nM, 88.6% of the sites possessed high affinity (pIC50 = 8.26) for alpha, beta-meATP. 2. The high affinity [3H]-alpha, beta-meATP binding sites appeared heterogeneous since in competition studies a number of nucleotide analogues (alpha, beta-meADP, ATP, ADP, AMP, GTP, GppNHp, GMP) and adenosine identified two populations of the sites labelled by 1 nM [3H]-alpha, beta-meATP. The proportion of sites with high affinity for these compounds was found to vary between 42 and 69%. 3. Approximately 60-69% of the binding sites labelled with 1 nM [3H]-alpha, beta-meATP possessed high affinity for alpha, beta-meADP (pIC50 = 8.87), AMP (pIC50 = 7.12), GMP (pIC50 = 7.34), UTP (pIC50 = 6.12), GTP (pIC50 = 7.59), GppNHp (pIC50 = 7.35) and adenosine (pIC50 = 5.45). The sites at which these compounds possessed high affinity were probably the same, since, in the presence of GMP at a concentration (10 microM) sufficient to inhibit selectively the binding of [3H]-alpha,beta-meATP, the [3H]-alpha,beta-meATP binding sites with high affinity for AMP, UTP, alpha, beta-meADP, GTP, GppNHp and adenosine were also occluded.4. WEC cell membranes were able to metabolize a trace concentration (6 nM) of [3H]-AMP to [3H]-adenosine under the conditions of the binding assay. The pIC50 values of adenosine (5.99), GMP (7.55)and the substrate AMP (7.19) for inhibiting this [3H]-AMPase activity were almost identical to their high affinity pIC50 estimates obtained in the binding assay. Although alpha, beta-meADP, alpha, beta-meATP, beta,upsilon-meATP,ATP, ADP and GppNHp identified heterogeneity in the [3H]-AMPase activity of the WEC cells, theirpIC50 values for inhibiting the major portion of the [3H]-AMPase activity were similar to their respective high affinity pIC50 values in the binding assay. It thus seems likely that WEC cells express a form of 5'-nucleotidase that possesses high affinity for both alpha,beta-meADP and alpha,beta-meATP and that this enzyme can be labelled by [3H]-alpha,beta-meATP.5. In the presence of 10 microM GMP, the affinity estimates for alpha,beta-meADP, AMP, GMP, GTP, GppNHp,ADP and adenosine at the high affinity [3H]-alpha,beta4-meATP binding sites that remained available, were lowa nd similar to their affinity estimates at the high affinity [3H]-alpha,beta-meATP binding sites of rat vas deferens. Since the high affinity [3H]-alpha,beta-meATP binding sites in rat vas deferens are thought to be P2x purinoceptors it is possible that the high affinity [3H]-alpha,beta-meATP binding sites in the WEC which possess low affinity for alpha,beta-meADP are also P2x purinoceptors.

5'-Nucleotidase↗
PubMed2000

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis.

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.

5'-Nucleotidase↗
PubMed1991

Inhibition of hexose monophosphate shunt in young erythrocytes by pyrimidine nucleotides in hereditary pyrimidine 5' nucleotidase deficiency.

Recent reports have suggested that haemolytic anaemia in pyrimidine 5' nucleotidase (P5'N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red-cell survival, we tested glucose 6-phosphate dehydrogenase (G-6PD), HMS, P5'N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5'N-deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both G-6PD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 mumol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red-cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5'N-deficient patients.

5'-Nucleotidase↗
PubMed2004

Expression of CD73/ecto-5'-nucleotidase on human gingival fibroblasts and contribution to the inhibition of interleukin-1alpha-induced granulocyte-macrophage colony stimulating factor production.

BACKGROUND AND OBJECTIVES: CD73/5'-nucleotidase (5'-NT) is an ectoenzyme that participates in immune/inflammatory reactions. We examined the possible expression of CD73/5'-NT on human gingival fibroblasts (hGF), which are important to the immune/inflammatory system in periodontal tissue. METHODS AND RESULTS: We demonstrated that CD73/5'-NT was expressed on hGF by flow cytometry. We found that pre-treatment of hGF with 5'-AMP induced marked inhibition of granulocyte-macrophage colony-stimulating factor (GM-CSF) production from hGF upon stimulation with interleukin-1alpha (IL-1alpha) by enzyme-linked immunosorbent assay (ELISA). A specific inhibitor of 5'-NT, adenosine 5'-[alpha,beta-methylene] diphosphate blocked the inhibition of GM-CSF production, suggesting that adenosine converted from 5'-AMP acts on the inhibitory effects. The GM-CSF inhibition suggested that A3 receptor might be involved. The rank order of agonists was found to be (N6-benzyl-5'-N-ethylcarboxamidoadenosine) A3 receptor agonist > or = (2-chloroadenosine) non-selective agonist > (CGS-21680) A2A receptor agonist > adenosine > or = (N6-cyclohexyladenosine) A1 agonist. Further support for the main role of A3 receptor was the binding A3 antagonist [9-chloro-2-(2-furanyl)-5-([phenylacetyl]amino)[1,2,4]-triazolo[1,5-c]quinazdine] reversed the effect of adenosine, but no significant reverse was observed by A1 (1,3-dipropyl-8-cyclopentylxanthine), A2 [3,7-dimethyl-1-(2-propargyl)xanthine], A2A[8-(3-chlorostyryl)caffeine], and A2B (alloxazine) antagonists. The CD73/5'-NT expression was increased upon stimulation with gamma-interferon, but not other stimulants such as tumor necrosis factor-alpha, IL-4, lipopolysaccharide from Porphyromonas gingivalis and Escherichia coli, and fimbriae from P. gingivalis, and this increase was correlated with the enhanced GM-CSF inhibition by 5'-AMP but not adenosine. CONCLUSIONS: These findings suggested that CD73/5'-NT on hGF exerts an anti-inflammatory effects in periodontal disease by conversion from 5'-AMP to adenosine.

5'-Nucleotidase↗
PubMed1990

The in vitro and in vivo effects of lead on delta-aminolevulinic acid dehydratase and pyrimidine 5'-nucleotidase.

The in vitro and in vivo effects of lead on the activity of pyrimidine-5'-nucleotidase (P5N, E. C. 3.1.3.5) and delta-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) were studied. Incubation of blood with lead at concentrations of up to 3 mumol/l (about 60 micrograms/dl) did not appear to affect the activity of P5N, while the activity of ALA-D decreased dose-dependently with lead. Administration of lead caused a marked and rapid suppression of ALA-D in rats. The suppression of lead on P5N appeared to be a rather slow process. The decrease of its activity only came into effect 20 days after administration with lead. these findings suggest that lead induced P5N inhibition is a slow process while the suppression of ALA-D activity occurs much earlier.

5'-Nucleotidase↗
PubMed2005

Expression of human ecto-5'-nucleotidase in pig endothelium increases adenosine production and protects from NK cell-mediated lysis.

Ecto-5'-nucleotidase (E5'N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. We evaluated whether expression of human E5'N on pig endothelial cells (EC) attenuates human NK cell-mediated cytotoxicity. A pig EC line was stably transfected with human E5'N and human NK cell adhesion and cytotoxicity toward pig EC cultures was measured by flow cytometry and intracellular enzyme release. E5'N activity in pig EC lysates increased from 0.68 +/- 0.07 to 1013 +/- 293 nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37 +/- 0.05 to >300 nmol/min/mg protein in non-transfected and transfected cells, respectively. The rate of adenosine production in transfected cells increased also with ATP as the extracellular substrate. Cytotoxicity of human NK cells was reduced from 10.7 +/- 0.4% and 11.1 +/- 1.1% with non-transfected pig EC to 5.2 +/- 0.2% and 5.0 +/- 0.2% in transfected cells with 50 microM and 250 microM AMP, respectively. Reduction of cytotoxicity in E5'N-transfected EC was abolished by the E5'N inhibitor and was mimicked in non-transfected EC by the addition of adenosine, demonstrating the key role of adenosine produced by E5'N in inhibiting NK cell cytotoxicity. We suggest that overexpression of E5'N in EC of transgenic pigs is a possible strategy to ameliorate rejection after xenotransplantation.

5'-Nucleotidase↗
PubMed2005

Adenosine deaminase and 5'-nucleotidase activities in saliva from patients with oral and laryngeal cancer.

OBJECTIVE: The aim of this study was to evaluate saliva's activities of adenosine deaminase (ADA) and 5'-nucleotidase (5'-NT) enzymes and their utility as diagnostic and therapeutic markers in oral and laryngeal cancer. MATERIALS AND METHODS: Pre- and post-operative saliva's activities of ADA and 5'-NT enzymes were measured in patients with squamous cell oral (n = 10) and laryngeal cancer (n = 17) and compared with control saliva samples (n = 19). RESULTS: The ADA was found to be lower in saliva of the patients with oral cancer compared with the laryngeal cancer and controls. However, no significant differences were found between pre- and post-operative values for both enzymes in the patient groups. We also could not find statistically significant differences between saliva's activities of 5'-NT in patients and control subjects. CONCLUSIONS: Low activity of ADA observed in saliva of the patients with oral cancer has been suggested as a compensatory mechanism against rapid purine and DNA metabolism in cancer cells. The current study does not support the hypothesis that saliva's activities of these enzymes may be used as additional diagnostic and prognostic cancer markers.

5'-Nucleotidase↗
PubMed1974

Ecto-enzyme of granulocytes: 5'-nucleotidase.

The 5'-nucleotidase of guinea pig polymorphonuclear leukocytes is localized exclusively on the plasma membrane of intact cells. The active site of this enzyme faces the external medium, not the cytoplasm.

Adenosine Monophosphate↗
PubMed1995

A salt-sensitive 3'(2'),5'-bisphosphate nucleotidase involved in sulfate activation.

Overexpression of a yeast gene, HAL2, allows the cells to tolerate higher than normal extracellular salt concentrations. HAL2 encodes a 3'(2')5'-bisphosphate nucleotidase that serves to remove the end products of sulfate transfer during cellular metabolism. The enzyme is inhibited by lithium and sodium and is activated by potassium. Metabolic systems that are sensitive to salt, as well as those governing osmolyte synthesis and ion transport, offer routes by which genetic engineering can be used to improve the tolerance of various organisms to salt.

Adenosine Diphosphate↗
PubMed2000

Identification of a Haemophilus influenzae 5'-nucleotidase protein: cloning of the nucA gene and immunogenicity and characterization of the NucA protein.

We report on the identification of a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, which elicits cross-reactive bactericidal antibodies against NTHi. The protein was extracted from NTHi strain P860295 with KSCN and purified; it migrated as a single band on a sodium dodecyl sulfate-polyacrylamide gel with an apparent molecular mass of 63 kDa. Mouse antiserum generated against the purified protein was reactive on whole-cell enzyme-linked immunosorbent assay (ELISA) with seven NTHi strains and type b Eagan and Whittier strains and exhibited bactericidal activity to homologous and heterologous NTHi strains. However, the protein is made in small amounts in NTHi as corroborated by immunoelectron microscopy. To further study this protein, we cloned, sequenced, and expressed it recombinantly in Escherichia coli. The recombinant protein is localized in the periplasm of E. coli and has been purified to homogeneity. Both the recombinant and native proteins possess 5'-nucleotidase activity; hence, the protein has been called NucA. Mouse antiserum directed against the recombinant NucA protein was reactive on Western immunoblots and whole-cell ELISA with all H. influenzae strains tested including Eagan and was bactericidal for two heterologous strains tested. The antiserum also resulted in a log reduction in bacteremia, in an infant-rat protection study with H. influenzae type b as the challenge strain. These features suggest that NucA is a potential subunit vaccine candidate against NTHi disease.

5'-Nucleotidase↗
PubMed1999

Reduced purine 5'-nucleotidase activity in lymphocytes of patients with systemic lupus erythematosus: results of a pilot study.

OBJECTIVE: To investigate purine metabolism in patients with systemic lupus erythematosus (SLE) for possible abnormalities that might be related to their overall impaired immune function. METHODS: This pilot study included 17 patients with SLE (2 men, 15 women). Enzyme activities of the purine enzymes 5'-nucleotidase (5'NT), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) were measured in peripheral blood mononuclear cells (PBMC) and also in fractions of T cells (differentiation antigen CD3+) (n = 12) and B cells (CD19+) (n = 9). The activity of the thiopurine enzyme thiopurine-methyltransferase (TPMT) was measured in red cell lysate. Routine blood tests and indices of disease activity were measured as well. Results were compared with those of healthy volunteers. RESULTS: Compared with their controls, the female SLE patients had a more than 50% reduced activity of 5'NT in the T cell fraction (p = 0.001) and in PBMC (p < 0.000). 5'NT activity was also lower in B cells, but this was not statistically significant. Enzyme activities did not correlate with indices of disease activity, disease duration or the B cell/T cell ratio and no influence of medication was found. CONCLUSION: Reduced lymphocyte 5'NT activity is a novel finding in SLE. These results indicate that purine metabolism in SLE may be disturbed. Consequences of a low 5'NT activity may be an intracellular accumulation of (deoxy)purine nucleotides and a reduction of adenosine production. It is hypothesised that these factors may play a part in the overall impaired immune function and in the chronicity of inflammation in SLE.

Adult↗