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PubMed1994

The bifunctional cytosolic 5'-nucleotidase: regulation of the phosphotransferase and nucleotidase activities.

The cytosolic 5'-nucleotidase specific for IMP, GMP, and their deoxyderivatives has been purified approximately 1000 times from calf thymus. The enzyme, in the presence of a suitable nucleoside, can act as a phosphotransferase, catalyzing the transfer of the phosphate moiety from a nucleoside monophosphate donor to a nucleoside acceptor, thus operating as an interconverting activity. This phosphorylating activity has drawn the attention of several research groups because the cytosolic 5'-nucleotidase represents the only cellular enzyme able to phosphorylate inosine and guanosine analogs, which are not substrates of known cellular nucleoside kinases. In this paper, we report the kinetic parameters of the bifunctional enzyme and its response to variations in adenylate energy charge. The results seem to indicate that in the presence of physiological concentrations of ATP and phosphate, the enzyme behaves mainly as a phosphotransferase, its activity being dependent only on the availability of a suitable nucleoside.

5'-Nucleotidase↗
PubMed1980

Cooperativity of the concanavalin A inhibition of bovine milk fat globule membrane 5'-nucleotidase. Response to extraction of nucleotidase and of putative cytoplasmic surface coat components.

5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) of bovine milk fat globules can be solubilized by deoxycholate from either isolated globule membranes or washed cream. The solubilized and membrane-bound enzymes exhibit similar Km values and are inhibited by concanavalin A by an apparent noncompetitive process. The soluble enzyme shows positive cooperativity for the inhibition (Hill coefficient of 2) at 37 degrees C, but the membrane enzyme exhibits essentially no cooperation effect. At lower temperatures (5 or 20 degrees C) the cooperative effect in the inhibition of the soluble enzyme is lost. Colchicine and cytochalasin D failed to induce cooperativity of the concanavalin A inhibition of the membrane enzyme, but induction cooperativity occurred when membranes were extracted with glycine/EDTA/mercaptoethanol, releasing a major protein component with a polypeptide molecular weight of 155 000. We suggest that the interaction of this component with the membrane imposes restraints on the behavior of the nucleotidase which are reflected in the cooperativity of the inhibition of the enzyme by concanavalin A.

Animals↗
PubMed2002

Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases.

The salivary apyrases of blood-feeding arthropods are nucleotide-hydrolyzing enzymes implicated in the inhibition of host platelet aggregation through the hydrolysis of extracellular adenosine diphosphate. A human cDNA homologous to the apyrase cDNA of the blood-feeding bed bug was identified, revealing an open reading frame encoding a 371-amino acid protein. A cleavable signal peptide generates a secreted protein of 333 residues with a predicted core molecular mass of 37,193 Da. Expression in COS-1 cells produced a secreted apyrase in the cell media. The ADPase and ATPase activities were dependent upon calcium, with a pH optimum between pH 6.2 and 7.2. Interestingly, the preferred substrate was not ADP, as might be expected for an enzyme modulating platelet aggregation, but rather UDP, followed by GDP, UTP, GTP, ADP, and ATP. The nucleotidase did not hydrolyze nucleoside monophosphates. Size-exclusion chromatography and Western blot analysis revealed a molecular mass of approximately 34-37 kDa. Treatment of the enzyme with peptide N-glycosidase F indicated that the protein is glycosylated. Northern analysis identified the transcript in a range of human tissues, including testis, placenta, prostate, and lung. No traditional apyrase-conserved regions or nucleotide-binding domains were identified in this human enzyme, indicating membership in a new family of extracellular nucleotidases.

Adenosine Triphosphatases↗
PubMed1986

Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia.

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5'-NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development.

5'-Nucleotidase↗
PubMed1981

Adenosine deaminase and ecto-5'-nucleotidase activities in various leukemias with special reference to blast crisis: significance of ecto-5'-nucleotidase in lymphoid blast crisis of chronic myeloid leukemia.

Adenosine deaminase (ADA) and ecto-5'-nucleotidase (5'-N) activities were examined in peripheral leukocytes from patients with leukemias, including nine patients with chronic myeloid leukemia (CML) in blast crisis. Four of none cases of CML in blast crisis were myeloid and the remaining lymphoid morphologically. The diagnosis of CML in lymphoid blast crisis was further contributed by the measurement of terminal deoxynucleotidyl transferase (TdT) activity. In all four cases of lymphoid blast crisis and one of myeloid blast crisis, leukemia cells had high 5'-N activity, while there was a little or no detectable activity in those from four cases of myeloid blast crisis and all of CML in chronic phase. ADA activity was high in seven of nine patients with blast crisis. Taken together, leukemia cells from two cases of lymphoid blast crisis had high ADA and 5'-N activities comparable to those in acute lymphocytic leukemia (ALL) cells. In contrast, the enzyme activities of leukemia cells from all but one patient in myeloid blast crisis were in a range similar to acute myeloid leukemia cells. The implications of these findings are as follows: (1) 5'-N may be used as a new biochemical marker of CML in lymphoid blast crisis. (2) Some lymphoid cells of CML in blast crisis have high ADA, 5'-N, and TdT activities and thus are very similar to ALL cells.

5'-Nucleotidase↗
PubMed1975

Studies on 5'-nucleotidase histochemistry. III. 5'-Nucleotidase activity in smooth muscle cells of the rat's gastrointestinal tube.

The distribution pattern of histochemically detectable 5'-nucleotidase (5'-Nase) activity is described in smooth muscle cells of the rat's gastrointestinal tube (esophagus, stomach, small intestine, large intestine). Both, light and electron microscopic methods are used. Faint positive 5'-Nase activity is observed on smooth muscle cells of the lamina muscularis mucosae in the thoracal esophagus whereas it is completely absent from smooth muscle cells of the abdominal esophagus and the stomach. In the small and large intestine strong positive 5'-Nase reaction is found on smooth muscle cells of the lamina muscularis mucosae and the innermost part of the lamina muscularis externa. In the circular and longitudinal layer of the lamina muscularis externa a slight increase in 5'-Nase activity is observed from the proximal to the distal segments. The reaction product is restricted to the outer cell surface of smooth muscle cells. In the small intestine the strong enzymatic activity in the innermost part of the muscularis externa is found to be localized at small and dense muscle cells (sd-cells). Common morphological and histochemical characteristics of sd-cells and smooth muscle cells of the lamina muscularis mucosae are emphasized. Hypothetical functions e.g. uptake of precursors of nucleosidephosphates, possible functional connection to a high glycogen content, correlation between 5'-Nase activity and proliferation capacity and local vasodilatory effect are discussed.

Adenosine Diphosphate↗
PubMed1996

No correlation between CD73 expression and ecto-5'-nucleotidase activity on blood mononuclear cells in vitro. Evidence of CD73 (ecto-5'-nucleotidase) on blood mononuclear cells with distinct antigenic properties.

Monoclonal antibodies against ecto-5'-nucleotidase (ecto-5'-NT) have been clustered to CD73. The aim of the present study was to elucidate whether specific ecto-5'-NT activity on blood mononuclear cells (BMC) was correlated with CD73 expression measured by flow cytometry. During culture of CD73-negative lymphocytes the percentage of cells with ecto-5'-NT activity increased without there being a comparable increase in CD73 expression. After 2 days' culture, 9% (3-16, median and range given) demonstrated ecto-5'-NT activity, whereas CD73-positive cells comprised only 1.6% (0.0-3.0). These results show the existence of lymphocytes with ecto-5'-NT activity but without expression of CD73. The ecto-5'-NT activity increased significantly (p<0.02) during culture of unseparated BMC, whereas the number of anti-CD73 binding sites did not change. As a consequence. the number of anti-CD73 binding sites per U ecto-5'-NT activity decreased during culture. Exposure to interleukin-4 or prostaglandin E2 changed the enzymatic activity but not the number of anti-CD73 binding sites. Treatment of BMC with phosphatidylinositol-specific phospholipase-C released 57% (51%-75%) of the ecto-5'-NT activity into the supernatants, without a detectable decrease in CD73 expression. The fact that ecto-5'-NT was removed from the supernatant after precipitation with anti-CD73 showed that the released ecto-5'-NT activity was due to enzymatic activity of CD73 molecules. The results of this study indicate that CD73 exists on BMC in isoforms with distinct capacities to bind mouse monoclonal anti-CD73.

5'-Nucleotidase↗