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PubMed1985

Ecto-5'-nucleotidase can provide the total purine requirements of mitogen-stimulated human T cells and rapidly dividing human B lymphoblastoid cells.

The ability of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells to drive their total purine requirements from inosine 5'-monophosphate, inosine, or hypoxanthine was compared. Inosine 5'-monophosphate first must be converted to inosine by the action of the enzyme ecto-5'-nucleotidase before it can be transported into the cell; inosine and hypoxanthine, however, can be transported directly. Mitogen-stimulated human peripheral blood T cells were treated with aminopterin to inhibit purine synthesis de novo and to make the cells dependent on an exogenous purine source. Thymidine was added as a source of pyrimidines. Under these conditions, 30 microM inosine 5'-monophosphate, inosine, and hypoxanthine showed comparable abilities to support [3H]thymidine incorporation into DNA or [3H]leucine incorporation into protein at rates equal to that of untreated control cultures. Similar results were found when azaserine was used to inhibit purine synthesis de novo, and thus DNA synthesis. In parallel experiments with the rapidly dividing human B lymphoblastoid cell line WI-L2, treatment with aminopterin (plus thymidine) inhibited the growth rate by greater than 95%. The normal growth rate was restored by the addition of 30 microM inosine 5'-monophosphate, inosine, or hypoxanthine to the medium. However, in similar experiments with cell line 1254, a derivative of WI-L2 which lacks detectable ecto-5'-nucleotidase activity, inosine and hypoxanthine (plus thymidine), but not inosine 5'-monophosphate (and thymidine) were able to restore the growth inhibition due to aminopterin. These results show that the catalytic activity of ecto-5'-nucleotidase is sufficient to meet the total purine requirements of mitogen-stimulated human T cells or rapidly dividing human B lymphoblastoid cells, and suggest that this enzyme may be important for purine salvage when rates of purine synthesis de novo are limited and/or an extracellular source of purine nucleotides is available.

5'-Nucleotidase↗
PubMed1985

Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation.

A cytoplasmic 5'-nucleotidase (EC 3.1.3.5) can catalyze the phosphorylation of inosine (Worku, Y., and Newby, A.C. (1982) Biochem. J. 205, 503-510). This enzyme was purified to determine whether it could catalyze the formation of trace levels of phosphorylated acyclovir (ACV), a nucleoside analog with antiherpes activity. Acyclovir phosphorylating activity from rat liver co-chromatographed with the enzyme throughout the 1200-fold purification and through size exclusion chromatography or polyacrylamide-gel electrophoresis. In addition, the pH optimum, ATP stimulation, and phosphate inhibition of the ACV phosphorylating activity paralleled those of the 5'-nucleotidase. Finally, ACV phosphorylation was competitively inhibited by inosine (Kis = 6.5 mM; K'm (inosine) = 5.0 mM). This was consistent with phosphorylation at a common catalytic site. In addition to inosine and ACV, the guanine derivatives Guo, dGuo, 9-beta-D-arabinofuranosylguanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were substrates for the enzyme. The relative phosphorylation rates were, respectively, 100, 0.7, 19, 4, 0.3, and 0.7, at 0.1 mM phosphate acceptor. Approximate K'm values were, respectively, 5, 90, 10, 10, greater than 100, and greater than 100 mM. Although the substrate activity of ACV with the 5'-nucleotidase was inefficient, it appeared to be sufficient to account for the small amounts of ACV phosphates formed in uninfected cells.

5'-Nucleotidase↗
PubMedDate not supplied

[Effect of hydrocortisone on the density-gradient distribution of autoreactive and 5'-nucleotidase-separating thymocytes].

Rat thymocytes forming rosettes with autologous erythrocytes, are separated by centrifugation in the ficoll-verografin density gradient into two unequal fractions. A minor part of rosette forming cells is found in the gradient layers with the density of 1.069 and 1.073, a major part in the layers with the density of 1.090 and 1.080. After the administration of hydrocortisone to rats (2.5 mg per 100 g of mass) the number of rosette forming cells increased but in the gradient light fraction (d = 1.069). At 37 degrees C this thymocyte fraction separates the greatest amount of protein with 5'-nucleotidase activity. Under hydrocortisone influence the number of cells in this fraction and the amount of separable protein per 10(6) cells as well as 5'-nucleotidase activity per 10(6) increased in this fraction only. It has been assumed that an increase in 5'-nucleotidase separated at 37 degrees C, is a more constant feature of cortisone resistant thymocytes than a low affinity to autologous erythrocytes which is usually regarded as a feature of mature thymocytes.

5'-Nucleotidase↗
PubMed1987

[Regression analysis in the study of dynamics of 5-nucleotidase activity in mouse peritoneal exudate macrophages].

The time course of the activity of 5-nucleotidase of peritoneal exudate macrophages (PEM) was studied on mice. Regression models of in time changes in the activity of PEM 5-nucleotidase after intraperitoneal administration of 2 immunostimulators: salmosan and blastolysine were constructed by the results of the study. Within the experimentally chosen periods of the study the time changes in the enzyme activity after administration of either salmosan or blastolysine could be adequately approximated by linear regression. The models enabled predicting the enzyme activity levels at various moments within wide ranges after administration of the immunostimulators. Moreover, it is recommended that the regression models of changes in 5-nucleotidase activity after intraperitoneal administration of the immunostimulators be used for quantitative comparison of their efficacy.

5'-Nucleotidase↗
PubMed1987

[Interstrain differences in changes in 5-nucleotidase activity in mouse macrophages in response to immunostimulation].

The effect of immunostimulants on the activity of 5-nucleotidase in the macrophages of peritoneal exudate (MPE) has been investigated in mice of various strains. It has been demonstrated that in case of subcutaneous introduction of immunostimulants interstrain differences might be observed in the changes of MPE 5-nucleotidase activity. The decrease in the enzymatic activity in MPE was found to be the most pronounced in C57Bl/6 mice, while in AKR mice it was the least marked. CBA and C3 mice revealed no changes in MPE 5-nucleotidase activity in response to immunostimulation.

5'-Nucleotidase↗
PubMed1984

[Role of adenylate kinase, AMP deaminase and 5'-nucleotidase in the metabolism of adenylic nucleotides].

The activities of adenylate kinase, AMP-deaminase and 5'-nucleotidase in various tissues of the rat were studied. The activity of the forward adenylate kinase reaction (ATP + AMP----2 ADP) against the back one (2 ADP----ATP + AMP) was predominant. The liver was shown to contain two, while the blood serum--three adenylate kinase isoenzymes. In the skeletal muscles, the catabolism of adenylic acid involving AMP-deaminase and 5'-nucleotidase predominantly occurred via deamination, in the liver--via dephosphorylation, while in the leucocytes, erythrocytes and blood serum the activity of these processes was essentially the same. In vitro, ATP enhanced the activity of AMP-deaminase in the liver, leucocytes and erythrocytes and decreased it in the blood serum. Under effects of ATP, the activity of 5'-nucleotidase in the leucocytes and blood serum was markedly elevated, that in the liver and erythrocytes was unaffected.

5'-Nucleotidase↗
PubMedDate not supplied

The effect of ethanol on 5'-nucleotidase of rat liver plasma membranes.

The activity of 5'-nucleotidase of rat liver plasma membranes has been investigated in normal and acutely ethanol-intoxicated rats (7 g ethanol/Kg body wt). Ethanol was also added to the incubation mixture for 5'-nucleotidase assay. The alcohol modified the Km of the enzyme when added to plasma membranes of normal rats; moreover, it increased the activation energy of the reaction. The treatment with the alcohol in vivo lowered the Vmax, but no modifications of Km could be detected in this case, upon further addition of the toxic in vitro. It is concluded that ethanol is able to act by itself on 5'-nucleotidase activity of rat liver plasma membranes; however, ethanol produces other effects in vivo, probably due to its metabolism.

5'-Nucleotidase↗
PubMed1981

[Early changes in retinal 5'-nucleotidase activity in hereditary retinal degeneration].

Activity of soluble cGMP phosphodiesterase (PDE) and of two membrane enzymes, 5'-nucleotidase and Na,K-ATPase, was studied in the developing retina of rats with inherited retinal degeneration. It was found that by day 10 of life, the content of 5'-nucleotidase in the afflicted rats was significantly reduced as compared with controls. This difference was unchanged throughout the subsequent animals' life. Na,K-ATPase activity in the afflicted and normal animals was the same. Within the first 45 days of life, PDE calculated with respect to the rhodopsin content was not different as regards both the afflicted and normal rats. When calculated with respect to protein, the changes in PDE corresponded with the reported data. The data obtained allowed a suggestion to be made that changes in 5'-nucleotidase in inherited retinal degeneration are disease-specific. They are accounted for by changes in the enzymes of nonphotoreceptor retinal membranes. The changes in PDE may be regarded as secondary, correlating with variation in the number of the photoreceptor membranes.

5'-Nucleotidase↗
PubMed1982

Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency: nucleotide inhibition of G6PD and the pentose phosphate shunt.

We evaluated the erythrocytes of two patients with hereditary pyrimidine 5'-nucleotidase deficiency. Significant findings included an increased reduced glutathione content, increased incubated Heinz body formation, a positive ascorbate cyanide test, and decreased intraerythrocytic pH. The pentose phosphate shunt activity of the patients' red cells as measured by the release of 14CO2 from 14C-1-glucose was decreased compared to high reticulocyte controls. Glucose-6-phosphate dehydrogenase (G6PD) activity in hemolysates from control erythrocytes was inhibited 43% by 5.5 mM cytidine 5'-triphosphate (CTP) and 50% by 5.5 mM in uridine 5'-triphosphate (UTP) at pH 7.1. CTP was a competitive inhibitor for G6P (Ki = 1.7 mM) and a noncompetitive inhibitor for NADP+ (Ki = 7.8 mM). Glutathione peroxidase, glutathione reductase, and 6-phosphogluconate dehydrogenase were not affected by these compounds. Pentose phosphate shunt activity in control red cell hemolysate at pH 7.1 was inhibited to a similar degree by 5.5 mM CTP or UTP. Since the intracellular concentrations of G6P and NADP+ are below their KmS for G6PD, these data suggest that high concentrations of pyrimidine 5'-nucleotides depress pentose phosphate shunt activity in pyrimidin 5'-nucleotidase deficiency. Thus, this impairment of the pentose phosphate pathway appears to contribute to the pathogenesis of hemolysis in pyrimidine 5'-nucleotidase deficiency hemolytic anemia.

5'-Nucleotidase↗
PubMedDate not supplied

[5-nucleotidase activity in rat leukocytes, erythrocytes and blood serum during radiation sickness].

A study of 5-nucleotidase activity in the formed elements and blood serum of rats 1, 3, 7, 15 and 30 days after X-irradiation has demonstrated that it was increased in leukocytes at early times and inhibited at later times of the development of radiation sickness. In erythrocytes, inhibition of 5-nucleotidase activity was most pronounced on the 3d day. In blood serum, the enzyme activity was virtually invariable. The addition of ATP to the incubation medium normalized 5-nucleotidase activity.

5'-Nucleotidase↗
PubMed1978

Ecto 5'-nucleotidase deficiency in primary hypogammaglobulinaemia.

The activity of the lymphocyte ectoenzyme 5'-nucleotidase is very low in the majority of patients with primary 'common variable' hypogammaglobulinaemia. In order to test whether this can be explained by lymphocyte subpopulation deficiencies we measured 5'-nucleotidase activity, using both biochemical and histochemical techniques, in purified T and B cells from patients and healthy subjects. Purified B cells from normal subjects have about four times the activity of T cells. This explains why the levels of lymphocyte 5'-nucleotidase activity are at the lower limit of the normal range in patients with X-linked hypogammaglobulinaemia who lack B cells. The low levels in the 'common variable' group can be explained by low activity in their T lymphocytes associated with either low activity in their B cells or depletion of B cells. The finding that inhibition of the enzyme does not interfere with in vitro lymphocyte transformation or immunoglobulin production in normal subjects indicates that the enzyme deficiency is not directly responsible for the hypogammaglobulinaemia. These and other studies suggest that this enzyme appears on lymphocytes at a certain stage of development and that both T and B lymphocytes in some patients with 'common variable' hypogammaglobulinaemia are developmentally immature.

Agammaglobulinemia↗
PubMed1992

5'-Nucleotidase immunoreactivity of perineuronal microglia responding to rat facial nerve axotomy.

The ecto-enzyme 5'-nucleotidase was localized immunocytochemically in the axotomized rat facial nucleus. As revealed by the monoclonal antibody 5N4-2,5'-nucleotidase immunoreactivity markedly increased on perineuronal microglia during the first week following axotomy, and gradually disappeared from these cells by the end of the third post-operative week. Interestingly, parenchymal microglia were not or only weakly stained. These findings indicate that 5'-nucleotidase 5N4-2-immunoreactivity may serve as a marker for perineuronal microglia, a population of satellite glial cells that appear to be actively engaged in lesion-induced synaptic changes during regeneration.

5'-Nucleotidase↗
PubMed1978

Effect of phosphate esters, nucleotides and nucleosides on 5'-nucleotidase of cultured mouse macrophages.

Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto-5'-nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5-fold increase in 5'-nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5-fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5'-nucleotidase. Other adenosine-containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction--adenosine--when added to the medium exhibited a toxic effect on these cells--as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5'-monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.

Adenine Nucleotides↗
PubMed1994

One-minute high-performance liquid chromatography assay for 5'-nucleotidase using a 20-mm reverse-phase column.

A general procedure is described for developing HPLC-based assays for nucleotidases by using a 20-mm C-18 reversed-phase column. The method is applied on the determination of pyrimidine 5'-nucleotidase activity in human erythrocytes lysate and plasma. The activity is detected using isocratic conditions which separate the product (nucleoside) from the substrate (nucleotide) in less than 1 min with 100 mM potassium phosphate, pH 6.0, at a 2 ml/min flow rate. Nucleoside products of the enzymatic reactions are directly quantitated from their ultraviolet absorbances. The activity value closely agreed with that determined by measuring the production of inorganic phosphate. The method does not require prior dialysis of the sample and is 50-fold more sensitive than that based on colorimetric measurements of inorganic phosphate. Furthermore, avoiding the use of radioactive materials offers significant advantages with respect to commonly adopted assays and can be conveniently used for the determination of nucleotidase activity from different sources.

5'-Nucleotidase↗
PubMed1997

Human seminal plasma soluble 5'-nucleotidase: regulatory aspects of the dephosphorylation of nucleoside 5'-monophosphates.

Human seminal plasma contains two enzyme activities both capable of dephosphorylating all nucleoside 5-monophosphates with different efficiency and specificity. Broad-spectrum soluble 5'-nucleotidase is the object of this paper which deals with the definition of the response of this enzyme to effectors, some physiological and others not naturally occurring. The enzyme did not show any product regulation as all the nucleosides tested caused a moderate effect on the hydrolysis of the substrates. Theophylline and other xanthine derivatives had no effect on enzyme activity, whereas glycerate 2,3-bisphosphate, like other soluble 5'-nucleotidases, caused a stimulation of the enzyme, especially toward CMP and UMP. 5-Deoxy-5-isobutylthiadenosine resulted in no inhibition of the hydrolysis of AMP and IMP. The enzyme was affected neither by monovanadate nor by decavanadate, whereas it was strongly inhibited by Ap5 A. Variations in adenylate energy charge did not cause any alteration of the enzyme activity toward AMP and only a slight decrease of the hydrolysis of IMP. These regulatory properties, distinct from those of other soluble 5'-nucleotidases, show that this form, newly isolated from human seminal plasma, is subject to an almost unique, tissue-specific regulation.

5'-Nucleotidase↗
PubMed1994

Ecto-ATPases and 5'-nucleotidases in the caveolae of smooth muscle. Enzyme-histochemical evidence may indicate a role for caveolae in neurotransmission.

We have demonstrated the localization of ecto-Ca-ATPase and 5'-nucleotidase activity in the caveolae of smooth muscle cells of guinea pig was deferens and the ileum longitudinal muscle strips with a cerium-precipitation enzyme-cytochemical method. The activities seemed to be strongest in the caveolae. Since the simultaneous presence of the 5'-nucleotidase activity supports the hypothesis that this ecto-Ca-ATPase activity does not have a pump function, but, together with 5'-nucleotidase, may play a role in neurotransmission, these specific membrane invaginations, the caveolae, have a functional relationship with transverse tubules of striated muscle.

5'-Nucleotidase↗
PubMed2001

E. coli 5'-nucleotidase undergoes a hinge-bending domain rotation resembling a ball-and-socket motion.

Structures of nine independent conformers of E. coli 5'-nucleotidase (5'-NT) have been analyzed using four different crystal forms. These data show that the two-domain protein undergoes an unusual 96 degrees hinge-bending domain rotation. Structures of the open and closed forms with substrates and inhibitors reveal that the substrate moves by approximately 25 A with the large domain rotation into the catalytic site. The domain motions derived from a comparison of the nine conformations agree well with motions obtained from a normal mode analysis in that all independent domain rotations are around axes that are roughly located in the plane which includes the domain centers and the hinge. Two residues, Lys355 and Gly356, form the core of the hinge region and undergo a large change of the main-chain torsion angles. The hinge-bending movement observed for 5'-nucleotidase differs markedly from a classical hinge-bending closure motion which involves an opening of the substrate or ligand-binding cleft between two domains. In contrast, the movement observed in 5'-nucleotidase resembles that of a ball-and-socket joint. The smaller C-terminal domain rotates approximately around its center such that the residues at the domain interface move in a sliding motion along the interface. Few direct interdomain contacts and a layer of water molecules between the two domains facilitate the sliding motion.

5'-Nucleotidase↗
PubMed1992

Isolation and characterization of the ecto-5'-nucleotidase from a rat glioblastoma cell line.

5'-Nucleotidase has been purified from rat glioblastoma cells (Rugli cells). The enzyme has been solubilized from plasma membranes by using Triton X-100 and CHAPS. Two affinity chromatographies on concanavalin A and 5'-AMP-Sepharose render the purified enzyme with a high specific activity (76.36 mumol AMP.min-1.mg-1). The purified enzyme gives a single polypeptide band on SDS-PAGE with an apparent molecular mass of 74 kDa. Active forms with an apparent molecular mass of 135 kDa and 268 kDa are observed when the purified enzyme is analyzed by gel filtration in the presence of either 0.6% sodium deoxycholate or 0.1% Triton X-100, respectively. The purified 5'-nucleotidase presents optimum activity at pH 7.8-8.1 either in the presence or in the absence of Mg2+. A linear Arrhenius plot is observed in the 25-46 degrees C temperature range and an activation energy of 33.7 KJ/mol is calculated. The enzyme is inhibited by EDTA; the activity is partially restored by different divalent cations as Zn2+, Mn2+, and Co2+. The hydrolysis of nucleosides 5'-monophosphate shows Michaelis kinetic. The enzyme is inhibited by nucleosides di- and triphosphate. 5'-Nucleotidase is a glycoprotein, being its activity inhibited at different extent by various lectins.

5'-Nucleotidase↗