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PubMed1984

Decreased 5'-nucleotidase activity in a T lymphocyte subpopulation from patients with B cell chronic lymphocytic leukemia.

The activity of the ectoenzyme 5'-nucleotidase (5'N) was determined in the T lymphocyte subpopulations from patients with chronic lymphocytic leukemia (CLL). 5'N could be detected only in the T cells from patients whose B cells also had enzyme activity. The specific activity of CLL T4 cells was 0.17 +/- 0.02 micron/h/mg protein, similar to that of normal T4 cells, which was 0.13 +/- 0.08 micron/h/mg. The CLL T8 cells, however, had a significantly lower 5'N activity (0.17 +/- 0.02 micron/h/mg) than normal T8 cells (0.41 +/- 0.11 micron/h/mg) (P = .003). Normal null cells had very low activity, while much higher levels were found in the null cells of CLL patients whose B cells had activity. These findings document a difference in activity of an enzyme between the T8 cell population of patients with CLL and that of normal subjects.

5'-Nucleotidase↗

PubMedDate not supplied

[Effect of ionizing radiation and physical loads on pyrimidine-5'-nucleotidase activity in rat liver subcellular fractions].

Activity of pyridine-5'-nucleotidase in the mitochondria and postmitochondrial supernatant of the rat liver has been studied as affected by the radiation and physical load (running till tiredness). It is shown that physical load leads to double increase of cytidinemonophosphatase activity and does not actually affect uridine monophosphate one. Irradiation evokes changes of cytidine monophosphatase activity of the studied fractions in time: 1 year after irradiation it increases by 45%, 1 day later it sharply changes and 3 days later it increases again. Mutual action of the irradiation and physical loading 1 year after the irradiation causes the decrease of uridine monophosphatase and cytidine monophosphatase activity and 3 days later their amount increases, especially in the postmitochondrial supernatant (2-53 times). Thus the data obtained show that the effect of the radiation and physical loading evoke rather essential and stable changes in the activity of key enzymes of uridine monophosphate and cytidine monophosphate.

5'-Nucleotidase↗

PubMed1996

Evidence for the involvement of ecto-5'-nucleotidase (CD73) in drug resistance.

Increased ecto-5'-nucleotidase (ecto-5'NT) protein expression in several multidrug-resistant (MDR) cell lines, documented previously by our group, suggests that this enzyme is involved in drug resistance. Here, Northern blot analysis of selected cell lines and their MDR variants positively correlated ecto-5'NT protein with its mRNA expression. An inhibitor of ecto-5'NT enzymatic activity, alpha,beta-methyleneadenosine 5'-diphosphate (AMP-CP), was used to determine if functionally active enzyme had a role in drug resistance. AMP-CP (0.3 mM) reversed the resistance of ecto-5'NT-positive MDR cells (MCF7/A6, L1210/A) to doxorubicin, whereas it did not affect the doxorubicin sensitivity of the ecto-5'NT-negative parental cell lines or that of 2 ecto-5'NT-negative MDR cell lines (HL60/VCR and A2780/DX5). Furthermore, AMP-CP increased rhodamine uptake and inhibited rhodamine efflux from ecto-5'NT-positive MDR cells without affecting ecto-5'NT-negative MDR cells. The presence of exogenous adenosine (0.5 microM) circumvented AMP-CP-induced inhibition of rhodamine efflux from EL4/ADM cells. AMP-CP inhibited the growth of the ecto-5'NT-positive L1210/A MDR cells but had no effect on the growth of the parental cell line. Determination of intracellular ATP levels indicated that MDR cells which had increased ecto-5'NT expression also had a lower intracellular ATP level than their parental cells. Our results suggest that, in certain MDR cell lines, ecto-5'NT serves as a required accessory molecule in resistance mediated by ATP-dependent mechanisms and that growth-sustaining nucleosides are provided by this salvage pathway.

5'-Nucleotidase↗

PubMed1990

Prognostic implication of ecto-5'-nucleotidase activity in acute lymphoblastic leukemia.

Ecto-5'-nucleotidase (5'-N) activity was determined in 191 patients (71 children and 120 adults) with acute leukemia. Elevated values for 5'-N were registered in common acute lymphoblastic leukemia (ALL), but blast cells of T-cell ALL (T-ALL) and common ALL antigen-negative non-T-ALL had low enzyme activity comparable with the values of acute non-lymphocytic leukemia. Dependence of remission duration on 5'-N activity was analyzed in 74 adults with ALL, treated similarly in a prospective multicenter trial. The remission curves for ALL patients with 5'-N activity lower than 10 nmol/h x 10(6) cells were substantially and significantly better than those of patients with high activity (greater than 10 nmol/h x 10(6) cells). This difference was also evident in the immunologic subclass common ALL. Statistical evaluation showed that an interaction between immunologic subtype of the blast cells and their 5'-N activity had prognostic significance for remission duration. In addition to the independent factor, initial age, this interaction was also prognostic for survival.

5'-Nucleotidase↗

PubMed1992

Adenosine levels in the postimplantation mouse uterus: quantitation by HPLC-fluorometric detection and spatiotemporal regulation by 5'-nucleotidase and adenosine deaminase.

Extracellular adenosine has the potential to influence many aspects of target cell metabolism. The present study has determined the endogenous levels of adenosine in the pregnant mouse uterus and developing embryo-decidual unit with respect to the expression of two key enzymes of adenosine metabolism, 5'-nucleotidase (5'-NT; EC 3.1.3.5) and adenosine deaminase (ADA; EC 3.5.4.4). To measure adenosine levels, nucleoside extracts were etheno-derivatized and quantitated by high-performance liquid chromatography-fluorescence detection (0.03 pmol/mg protein sensitivity). Adenosine levels were determined to be 0.18 nmol/mg protein in the nonpregnant uterus; however, two statistically significant changes were identified in the pregnant uterus: (1) a periimplantation surge between day 3 (0.24 nmol/mg protein) and day 5 (0.59 nmol/mg protein) of gestation (plug day 0; implantation day 4); and (2) an early postimplantation decline between day 6 (0.54 nmol/mg protein) and day 7 (0.10 nmol/mg protein). The periimplantation adenosine surge coincided with uterine expression of 5'-NT, an enzyme which catalyzes the irreversible dephosphorylation of 5'-AMP to adenosine. 5'-NT expression was shown by Northern blot analysis to peak in the embryo-decidual unit on day 5 of gestation and then to decline through day 9; transcripts remained elevated in the placenta between day 9 and day 13 (the latest day examined in this study). By use of specific enzyme histochemistry, most 5'-NT activity was localized to the primary decidual zone on day 5. This expression subsequently declined during regression of the primary decidua; however, 5'-NT appeared on giant trophoblast (days 7-13) and the metrial gland (days 11-13). Other purine catabolic enzymes degrading AMP (adenylate deaminase) or generating adenosine (S-adenosylhomocysteine hydrolase) were not detected in the embryo-decidual unit suggesting that the net flux of utero-placental AMP catabolism proceeds with adenosine as an intermediate, this being the major pathway of adenosine formation. The sharp drop in adenosine levels between day 6 and day 7 coincided with a rise in the activity and mRNA expression of ADA, an enzyme which catalyzes the irreversible deamination of adenosine to inosine. ADA was previously localized to the secondary decidual zone (days 6-11), secondary giant cells (days 7-13), and spongiotrophoblasts (days 8-13) in the mouse (Knudsen et al., 1991). Results of developmental Northern blot analysis demonstrated a direct correlation of relative 5'-NT/ADA mRNA band intensity to adenosine content between day 4 and day 9 of gestation, suggesting that the local availability of adenosine in the antimesometrium is dependent upon the distribution of these enzymatic activities.(ABSTRACT TRUNCATED AT 400 WORDS)

5'-Nucleotidase↗

PubMed1990

5' Nucleotidase in chronic B cell leukemias: a cytochemical and ultrastructural study.

We studied by cytochemical means the distribution of 5' nucleotidase (5' NT), a purine degradative enzyme, in the circulating lymphocytes of 24 healthy donors and 41 cases of chronic lymphoid leukemias, classified according to morphological and immunological criteria. About half the normal circulating lymphocytes were 5'NT positive and exhibited variable degrees of enzyme activity. Among chronic B lymphocytic leukemias we found high percentages of positive cells only in the phenotypically more mature cases. Moreover all cases of hairy cell, follicular cell, lymphoplasmacytic, and plasma cell leukemia showed moderate or weak 5' NT reactivity. Also one case of chronic T lymphocytic leukemia, CD8 positive, was moderately positive, while another, with large granular lymphocyte morphology, was completely negative. Electron microscopy revealed a discontinuous, granular plasma membrane reaction pattern, varying in intensity from case to case. In conclusion, our results confirm the usefulness of the 5' NT cytochemical reaction for identification of lymphoid populations at different stages of maturation in chronic B cell disorders.

5'-Nucleotidase↗

PubMed1991

Synovial fluid 5'-nucleotidase activity. Relationship to other purine catabolic enzymes and to arthropathies associated with calcium crystal deposition.

We measured 5'-nucleotidase (5NT) activity in synovial fluid from 159 patients with various diagnoses. The activity of 5NT was compared with activities of nucleotide pyrophosphohydrolase, alkaline and neutral phosphatases, and adenosine deaminase, in the same samples. Higher levels of 5NT activity occurred in synovial fluid from osteoarthritic joints than from joints of patients with gout, pseudogout, or rheumatoid arthritis. The highest levels of 5NT activity were found in synovial fluid from patients with Milwaukee shoulder syndrome and from osteoarthritis patients in whom deposition of calcium-containing crystals was also present.

5'-Nucleotidase↗

PubMed1993

Human erythrocyte pyrimidine 5'-nucleotidase isoenzymes: effect of sulfhydryl reagents and electrophoretic discrimination.

The electrophoretic behavior of human red cell pyrimidine 5'-nucleotidase isozymes (UMPH1 and UMPH2) was studied on starch gels with and without treatment with thiol reagents. It was found that at least one reactive sulfhydryl group occurs in the UMPH1 isozyme but not in the UMPH2 isozyme. An electrophoretic system is described that allows the discrimination of UMPH1 and UMPH2 isozymes.

5'-Nucleotidase↗

PubMed2003

Analysis of 5' nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium.

In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.

5'-Nucleotidase↗

PubMed1994

Ecto-5'-nucleotidase (CD73) in multidrug-resistant cell lines generated by doxorubicin.

Cytochemical screening for a panel of enzymes revealed increased 5' nucleotidase (5'NT) expression in 3 of 3 P-glycoprotein 170 (Pgp170)-positive multidrug-resistant (MDR) variants of the murine EL4 T-lymphoma cell line (EL4/ADM, ER2 and ER13). Electron microscopic localization established the presence of the membrane-bound ecto-form of the enzyme. Nine other murine, human and Chinese hamster cell lines and their MDR variants were tested for ecto-5'NT. Of these, 4 MDR variants (human cell lines MCF7A6, MCF7A2, HeLaJ2C and the murine cell line L1210A) showed increased expression of ecto-5'NT, when compared with their parental cell lines. The findings with cells of human origin were confirmed by immunofluorescent localization with a specific monoclonal antibody (MAb) (27.2) against the human ecto-5'NT. All MDR cell lines with elevated ecto-5'NT expression were generated by doxorubicin treatment. These cells were more sensitive than their parental cell lines to AMP at concentrations of 1.5-3.0 mM, confirming that the expressed ecto-5'NT was biologically active. The parental and MDR cells did not differ, in general, in their sensitivity to adenosine. An inhibitor of ecto-5'NT, alpha,beta-methyleneadenosine 5'-diphosphate, completely reversed the resistance of the EL4/ADM cell line to doxorubicin. The possibility exists of a functional relationship between the ecto-5'NT molecule and the members of the ATP-binding cassette transporter superfamily, important components of MDR, in some cell types.

5'-Nucleotidase↗

PubMed1986

Erythrocyte pyrimidine 5'-nucleotidase inhibition by acute lead exposure in neonatal rats.

Inhibition by lead of erythrocyte pyrimidine 5'-nucleotidase (P5N) is thought to contribute to morphological abnormalities observed in red blood cells (RBC) of lead-exposed subjects. However, neither the mechanism of lead inhibition of P5N nor the relationship of this inhibition to blood lead levels attained in exposed subjects is known. In the present investigation, acute in vivo and in vitro lead acetate effects on erythrocyte P5N from 21-day-old rat pups were determined and were related to blood lead concentrations ascertained by atomic absorption spectrophotometry. Acute lead administration to rat pups resulted in a 16% to 21% reduction in erythrocyte P5N, with mean blood lead levels ranging from 77 to 108 micrograms/dl 24 hours later. Inhibition of erythrocyte P5N was linearly related to blood lead level (r = -0.67, P less than 0.05) following acute lead administration. Lead acetate addition to RBC preparations from 21-day-old rats resulted in concentration-dependent P5N inhibition which was comparable to that produced following acute in vivo exposure. The results indicate that acute P5N inhibition in lead-treated neonatal rats is due to noncompetitive P5N inhibition by lead. The inhibition of P5N produced by acute lead treatment is linearly related to blood lead concentrations.

5'-Nucleotidase↗

PubMed2004

Effects of starvation on haemolymphatic glucose levels, glycogen contents and nucleotidase activities in different tissues of Helix aspersa (Müller, 1774) (Mollusca, Gastropoda).

In the present study, the glucose concentration in the haemolymph and glycogen levels were determined in the various body parts of the Helix aspersa snail after feeding lettuce ad libitum and after various periods of starvation. To characterize the effect of starvation on nucleotidase activity, enzyme assays were performed on membranes of the nervous ganglia and digestive gland. Results demonstrated the maintenance of the haemolymph glucose concentration for up to 30 days of starvation, probably due to the consumption of glycogen from the mantle. In the nervous ganglia, depletion of glycogen occurs progressively during the different periods of starvation. No significant changes were observed on ATP and ADP hydrolysis in the membranes of nervous ganglia and no alterations in Ca2+ -ATPase and Mg2+ -ATPase occurred in the membranes of the digestive gland of H. aspersa during the different periods of starvation. Although there were no changes in the enzyme activities during starvation, they could be modulated by effectors in situ with concomitant changes in products/reactants during starvation.

Adenosine Triphosphatases↗

PubMed2001

Identification of inactive ecto-5'-nucleotidase in normal mouse muscle and its increased activity in dystrophic Lama2(dy) mice.

Ecto-5'-nucleotidase (eNT) activity and protein in normal (NM) and merosin-deficient dystrophic (DM) Lama2(dy) mice muscle were studied. eNT activity in DM was three- to four-fold that in NM. eNT in NM and DM displayed the same kinetic properties. Slot and Western blotting revealed that the immunoreactive protein was two to three times more abundant in control muscle, when NM and DM samples with the same eNT activity were compared, indicating that mouse muscle contains catalytically inactive eNT components. eNT activity and protein peaks coincided in sedimentation analyses, revealing that inactive eNT occurs as dimers. Most eNT activity and protein of NM bound to Lens culinaris (LCA) or Ricinus communis (RCA) agglutinins, but half of the activity and one-third of the protein bound to wheat germ agglutinin (WGA). Although WGA interaction did not permit full separation of inactive eNT, the results suggest that similar proportions of active species with and without WGA reactivity occur in mouse muscle, whereas a great fraction of the inactive eNT variants lack WGA reactivity. Because the level of eNT protein was little modified in DM, the higher eNT activity in dystrophic than in control muscle may result from misregulation in the synthesis of active and inactive eNT species or from conversion of inactive into active components.

5'-Nucleotidase↗

PubMed2005

Molecular cloning and biochemical characterization of a 3'(2'),5'-bisphosphate nucleotidase from Debaryomyces hansenii.

The enzyme 3'(2'),5'-bisphosphate nucleotidase catalyses a reaction that converts 3'-phosphoadenosine-5'-phosphate (PAP) to adenosine-5'-phosphate (AMP) and inorganic phosphate (Pi). The enzyme from Saccharomyces cerevisiae is highly sensitive to sodium and lithium and is thus considered to be the in vivo target of salt toxicity in yeast. In S. cerevisiae, the HAL2 gene encodes this enzyme. We have cloned a homologous gene, DHAL2, from the halotolerant yeast Debaryomyces hansenii. DNA sequencing of this clone revealed a 1260 bp open reading frame (ORF) that putatively encoded a protein of 420 amino acid residues. S. cerevisiae transformed with DHAL2 gene displayed higher halotolerance. Biochemical studies showed that recombinant Dhal2p could efficiently utilize PAP (K(m)17 microM) and PAPS (K(m)48 microM) as substrate. Moreover, we present evidence that, in comparison to other homologues from yeast, Dhal2p displays significantly higher resistance towards lithium and sodium ions.

Adenosine Diphosphate↗

PubMed2002

Human erythrocyte pyrimidine 5'-nucleotidase, PN-I.

Erythrocyte maturation is accompanied by RNA degradation and release of mononucleotides. Pyrimidine 5'-nucleotidase, PN-I, has been purified and characterized. The molecular and enzymatic properties determined for the enzyme shows a 36-kDa and 5.1 pI monomeric protein with no disulfide bridges and no phosphate content. The activity is dependent on Mg(2+), while it is inactivated by heavy metals and by thiol-reactive reagents. PN-I is specific for pyrimidine nucleoside monophosphates, including the antineoplastic agents 5'-AZTMP and 5'-Ara-CMP. PN-I possess phosphotransferase activity able to exchange phosphate between pyrimidine nucleoside monophosphates and pyrimidine nucleosides, including AZT and Ara-Cyd. Amino acid sequence has been obtained from tryptic and CNBr peptides. PN-I cDNA sequence, coding for a 286-residue protein, has been retrieved from tag database, amplified by PCR, and expressed in Escherichia coli. The recombinant protein was fully active and showed identical properties with respect to PN-I. Substantial identity has been revealed with the partial sequences reported for p36, an alpha-interferon-induced protein. The significance of this identity is discussed.

5'-Nucleotidase↗

PubMed1999

Microassay of 5'-nucleotidase and adenosine deaminase activity in microdissected nephron segments.

The present study describes a new method for microassay of the activity of 5'-nucleotidase (5'-ND) and adenosine deaminase (ADA) in the microdissected nephron segments. The nephron segments including glomeruli, proximal convoluted and straight tubules (PCT and PST), cortical and medullary thick ascending limbs, and cortical and medullary collecting ducts were microdissected. 5'-ND and ADA in the nondenatured lysate of 20-mm microdissected tubules and 20 glomeruli were separated by agarose gel electrophoresis and by isoelectric focusing, respectively. The gels were incubated with specific substrates and staining dyes to exhibit the dephosphorylation by 5'-ND or deamination by ADA. The enzyme activity was estimated by measuring the intensity of the reaction bands on the gels. The 5'-ND activity was detected in all microdissected tubular segments and glomeruli. Among these nephron segments, PCT and PST exhibited the greatest enzyme activity, averaging 1142 and 939 mU/mg tissue protein, respectively. The activity of ADA was also detected in all tubular segments and glomeruli. However, the greatest activity of this enzyme was found in the glomeruli (649.8 mU/mg protein). Using reverse transcriptase-polymerase chain reaction technique, we verified the presence of mRNA of 5'-ND and ADA in all microdissected tubular segments and glomeruli. Based on these results, we conclude that 5'-ND and ADA are present in all nephron segments studied, but the activity of these enzymes is nonuniformly expressed along the nephron. This microassay is a highly specific, sensitive, and reliable method for the segmental analysis of adenosine metabolism in the kidney.

5'-Nucleotidase↗

PubMed1996

The isolation from human seminal plasma of a new form of soluble 5'-nucleotidase.

Soluble broad spectrum 5'-nucleotidase from human seminal plasma was purified to homogeneity by a combination of (NH4)2SO4 precipitation, affinity chromatography, and gel filtration. The pure enzyme had a specific activity of 4800 nmol min-1 mg-1. SDS-PAGE of purified enzyme preparation revealed a single polypeptide band of 53 kDa and a tetrameric structure of 203 kDa was proposed for the native enzyme. This form had modest preference for AMP as substrate; Mg2+ and Mn2+ were activators of the enzyme although its activity was not absolutely dependent on the presence of these exogenous bivalent cations. The enzyme, recovered in the nonsedimentable fraction of human seminal plasma, had a pH optimum of 7.5; ATP and ADP were inhibitors of mixed type, Pi was a potent inhibitor at nonphysiological concentrations, and Con A and adenosine 5-[alpha, beta-methylene]diphosphate had no effect on the enzyme activity. The enzyme described here therefore has some unique properties between truly cytoplasmic and membrane-bound derived forms.

Adenosine↗

PubMed1996

Effect of various types of exercise training on 5'-nucleotidase and adenosine deaminase activities in rat heart: influence of a single bout of endurance exercise.

The aim of the present study was to find out whether activities of the enzymes controlling adenosine metabolism, 5'-nucleotidase (5NT) and adenosine deaminase (ADA), in the left ventricle of the rat's heart change after 6 weeks of endurance or sprint training. Additionally, an influence of a single bout of endurance exercise till exhaustion on activities of these enzymes was investigated in sedentary and trained rats. The rats were divided into three groups: (1) sedentary controls (C), (2) endurance-trained (ET), and (3) sprint-trained (ST). It was shown that both types of training increased 5NT, but did not change ADA activity in the rat heart. Acute exercise till exhaustion did not affect 5NT activity in the heart taken from C and ST rats, but decreased its activity in the ET group. The heart ADA activity after exhaustive exercise increased in C and in ET group, but decreased in ST animals. It is concluded that physical training affects cardiac adenosine metabolism and the type of training may exert an influence on purine nucleotides metabolism in the heart during exhaustive exercise.

5'-Nucleotidase↗