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PubMed1989

Ecto-5'-nucleotidase: localization in rat kidney by light microscopic histochemical and immunohistochemical methods.

We demonstrated the distribution pattern of ecto-5'-nucleotidase (5'-Nu) in rat kidney by enzymatic activity (lead salt precipitation) and by immunohistochemistry with a polyclonal antibody raised in rabbits. Enzyme activity was found in the brush border of the proximal tubule, highest in the P1 segments with decreasing intensity in the P2 segments and weakest in P3 segments in the medullary rays of the cortex. The P3 segments of the outer stripe showed slightly higher activity. Activity was also apparent in the intercalated cells in the connecting tubule and collecting duct, whereas all other tubular and glomerular structures were negative. Activity in peritubular and perivascular connective tissue was highest in the cortical labyrinth, weak or absent in the medullary rays of the cortex, and entirely absent in the medulla. The distribution of the antigen was fully congruent with that of the enzyme activity. With respect to the role of adenosine in regulation of renal blood flow and glomerular filtration rate, the distribution of 5'-Nu in the cortical interstitium may be particularly significant. The possibility of nucleotide cleavage at the brush-border membranes may be important for salvage of nucleotides from the tubular lumen.

5'-Nucleotidase↗
PubMed1983

5'nucleotidase activity in mitogen-stimulated adult and cord blood lymphocyte cultures.

Ecto 5'Nucleotidase (5'N) activity was determined in adult and cord blood lymphocytes before and after stimulation by phytohemagglutin (PHA) and pokeweed mitogen (PWM). After 7 days of culture, the mean 5'N level +/- S.E. in unstimulated adult cells was 15.9 +/- 3.21 nm/h/10(6) cells. Cells stimulated by PHA and PWM for 7 days had 5'N levels of 44.65 +/- 6.69 nm/h/10(6) cells and 33.24 +/- 7.67 nm/h/10(6) cells, respectively. Unstimulated cord blood cells cultured for 7 days had a mean 5'N level of 3.22 +/- 0.94 nm/h/10(6) cells whereas the level in PHA-stimulated cultures was 39.50 +/- 10.52 nm/h/10(6) cells and in PWM stimulated cultures 46.01 +/- 8.93 nm/h/10(6) cells. T-cell enriched and T-cell depleted subpopulations stimulated with mitogen generally did not show as great a degree of enhancement of 5'N activity as did unfractionated cells. This study indicates that 5'N activity is enhanced in cells that have been activated by mitogen stimulation. Although unstimulated cord blood cells have lower levels of 5'N activity than adult cells they have the capability upon stimulation to express levels equal to those found in adults.

5'-Nucleotidase↗
PubMed1981

Selective binding of prostaglandin F2 alpha to membrane fractions enriched in 5-nucleotidase, Ca2+-ATPase, and Mg2+-(Na+ + K+)ATPase.

Homogenates of uterine smooth muscle of laying hens were fractionated by differential centrifugation. Three of the five fractions thus obtained were further separated on sucrose gradient into four subfractions. By employing various enzyme markers, such as 5'-nucleotidase, Mg2+-(Na+ + K+)-ATPase, alkaline phosphatase, Ca2+-ATPase, cytochrome oxidase, acid phosphatase, and N-beta-acetyl glucosaminidase, the major subcellular fractions have been tentatively identified. Using gel filtration to separate bound and free prostaglandin F2 alpha (PGF2 alpha) we found that the specific uptake of PGF2 alpha was highest in the subfractions which also exhibited the highest enrichment in enzymes viewed generally as markers for cell membrane. The results suggest that PGF2 alpha-induced contractile activity in this tissue is initiated by the specific interaction of this agonist with discreet receptors in the sarcolemma, rather than by binding of PGF2 alpha to intracellular organelles or to a cytosolic receptor.

5'-Nucleotidase↗
PubMed1987

Effect of concanavalin A on 5'-nucleotidase activity of rabbit blood platelets.

Ecto-5'-nucleotidase (ecto-5'-NU) of platelets was enhanced by concanavalin A (Con A). This effect of Con A was antagonized by alpha-methyl-D-mannose, a specific antagonist of Con A binding to glycoprotein. Coformycin, an adenosine deaminase inhibitor, did not change the effect of Con A on the ecto-5'-NU. Uptake of adenosine by platelets was not affected by Con A. It was suggested that the ecto-5'-NU of platelet might be a direct and primary site of action of Con A.

5'-Nucleotidase↗
PubMed1980

Automated method for the determination of 5'-nucleotidase in serum by continuous flow analysis.

The manual method of Persijn & Van der Slik (J. Clin. Chem. Clin. Biochem. 6, 441-446 (1968): 7, 493-497 (1969); 8, 398-402 (1970) for the determination of serum 5'-nucleotidase has been adapted to the Auto-Analyzer II System. In the Auto-Analyzer II, the incubation temperature for the enzyme reactions is 37 degrees C, and the effective sample speed is 30/h, at a sample/wash ratio of 2:1. There is good agreement and correlation between the manual method and the Auto-Analyzer II method (equation of regression line: y = x + 0.6, correlation coefficient = 0.988; the normal range is 2.9-10.5 U/l for both methods). In routine use, within-run and between-day reproducibility are considerably smaller for the Auto-Analyzer II than for the manual method, especially when large numbers of samples are assayed.

5'-Nucleotidase↗
PubMed1983

Continuous assay of serum 5-nucleotidase activity with inosine 5'-monophosphate as substrate and total automation using a transfer-analyzer (Kem-O-Mat).

The continuous spectrophotometric assay of 5'-nucleotidase originally described by Heinz et al. ((1980) J. Clin. Chem. Clin. Biochem. 18, 781-788) was modified and fully automated on a Kem-O-Mat transfer analyzer, using inosine 5'-monophosphate as substrate. The reaction product was hydrogen peroxide and the reduction of NADP was observed for 10 minutes at 340 nm and at a reaction temperature of 30 degrees C. The different factors involved in the enzyme reaction were checked, including the substrate concentration, reaction rate, linearity and substrate preservation. Normal values ranged from 1 to 13 U/l. Between-day reproducibility was estimated with two different commercial control sera, and the coefficient of variation was 5% for the upper limit of normal activity (23 U/l). There was good agreement between the present method and a semi-automatic colorimetric technique (for 100 sera tested by both methods, the correlation coefficient was 0.974 and the regression line equation, y = 0.85 x- 1.5). Despite the lengthy reagent mixture preparation procedure, the method permitted assay of 50 samples per hour. The occurrence of high serum blanks in certain pathological states is discussed.

5'-Nucleotidase↗
PubMedDate not supplied

A study of the substrate and inhibitor specificities of AMP aminohydrolase, 5'-nucleotidase, and adenylate kinase with adenosine carboxylates of variable chain length.

A series of AMP analogs in which a terminal carboxylate residue, linked to C 4' of the ribose moiety of adenosine by zero, one, or two methylene groups (1,2,3) or by the unsaturated ethylidene link (4) replaces the phosphate anion, is tested for activity as substrates or effectors of three enzymes known to interact with AMP with a different degree of specificity. 2-4 are substrates of AMP aminohydrolase, 3 and 4 are competitive inhibitors of adenylate kinase, and all acids produce competitive inhibition of the least specific enzyme, 5'-nucleotidase. These activities can be correlated with the intramolecular flexibility of anionic substituent and adenine base which in turn is expressed in typical shifts of the proton magnetic resonance signal of purine H-8. The uronic acid 1, having a rigid molecular conformation, is inactive towards two AMP-dependent enzymes and little active with the third, indicating that this type of compound is not suitable as a nucleotide antagonist whereas nucleoside carboxylates of type 2 and 3 have a higher potential as effectors of nucleotide metabolism.

5'-Nucleotidase↗
PubMed1983

Ecto-5'-nucleotidase activities in B lymphoblastoid cell lines at various stages of maturation.

B lymphoblastoid cell lines (LCL) at various stages of maturation, including possible precursor B cell lines without immunoglobulin (Ig) expression, pre-B cell-like LCL, and Ig-secreting LCL, were established from peripheral and bone marrow mononuclear cells of four patients with congenital agammaglobulinemia (CAG) by infection with Epstein-Barr virus (EBV) and estimated for their ecto-5'-nucleotidase (ecto-5'-NT, E.C. 3.1.3.5) activity. Ecto-5'-NT activities in Ig-secreting LCL derived from CAG patients fell into the same range of those in LCL derived from control subjects (from 7.3 to 33.0 nmoles/hr/10(6) cells). When ecto-5'-NT activities of precursor B cell lines derived from the CAG patients were also compared with Ig-secreting LCL derived from control subjects, no significant difference in ecto-5'-NT activities was found among LCL with various Ig expression which might indicate various stages of B cell maturation. These results may suggest that ecto-5'-NT does not appear to be a biochemical marker of B cell maturation.

5'-Nucleotidase↗
PubMed1984

Decreased ecto-5'-nucleotidase activity in rabbit lymphocytes during experimental lens-induced uveitis.

An experimental uveitis was produced by immunizing albino and pigmented rabbits with heterologous (rat) lens protein six times at two-week intervals. After sensitization the anterior lens capsules were disrupted in an operation. After operation the rabbits were examined repeatedly with a slit-lamp. On the seventh day after operation the animals were sacrificed and both eyes were enucleated. The inflammatory reaction in the uveal tract was verified histopathologically. Thymus and spleen lymphocytes were purified using BSA-gradients. Ecto-5'-nucleotidase (AMP-ase) activity on the intact lymphocytes was measured biochemically and B-cells were counted using the fluorescent anti-IgG technique. Purified spleen and thymus lymphocytes were also analyzed using transmission electron microscopy. The AMP-ase activity of purified spleen lymphocytes was significantly decreased in albino rabbits suffering from experimental lens-induced uveitis when compared to that observed in control animals. The AMP-ase activity was also decreased in pigmented rabbits suffering from a very mild uveitis. Moreover, a decreased AMP-ase activity was observed in albino rabbits which were only immunized but not operated on and did not show any signs of uveitis. Differences in neither the B-cell proportions nor the ultrastructure of the purified lymphocytes could be observed in any of these experimental groups. It is proposed that the biochemical change in spleen lymphocytes observed in the present work is a reflection of the general immunological state which in certain conditions may lead to uveitis.

5'-Nucleotidase↗
PubMed1980

Pyrimidine 5'-nucleotidase deficiency: studies of five cases in two Japanese families.

Two new families with red cell pyrimidine 5'-nucleotidase (P5N) deficiencies were found in Japan. The enzyme activities were 4.8% in case 1 and 9.7% in case 2. The propositi showed characteristic hemolytic anemia with market basophilic stippling, increased reduced glutathione content and accumulation of pyrimidine nucleotides in the red cell. High Michaelis constants for cytidine monophosphate, normal thermostability, abnormal optimum pH in both cases, and normal electrophoretic mobility in case 1 and slower mobility in case 2 were shown, suggesting that the cause of P5N deficiency, at least in these cases, is due to the structural gene mutation.

5'-Nucleotidase↗
PubMed1987

Redistribution of Mn++-dependent pyrimidine 5'-nucleotidase (MDPNase) activity during shedding and phagocytosis.

Ultrastructural cytochemistry was used to localize a previously undescribed retinal enzyme activity, ie, "manganese-dependent pyrimidine 5'-nucleotidase" (MDPNase) activity in retinas from rats raised in cyclic light and killed at various times in the lighting cycle. In retinas fixed during the last 3 hr of darkness, at the beginning of the shedding period, heavy cytochemical staining was observed over the extracellular surfaces of the apical processes of the retinal pigment epithelium (RPE) cells. The adjacent distal ends of the rod outer segments (ROS), ensheathed by the apical processes, were also stained along their surfaces. During the first 3 hr after light onset, at the time of maximal shedding and phagocytic activity, the tips of the ROS and ROS phagosomes were heavily labeled with reaction product distributed throughout the interdisc spaces. The RPE apical processes, previously heavily stained, were at this time weakly reactive relative to the ROS tips and phagosomes. After the shedding peak (4-8 hr after light onset), the tips of the ROS were no longer labeled with reaction product, and the apical processes were unreactive. The proximal portions of the ROS were weakly stained throughout the lighting cycle. The observed patterns of redistribution of MDPNase activity before, during, and after the shedding peak suggest that the presence of the enzyme in the ROS tips may be correlated with shedding. Changes in the staining of the RPE apical processes in relation to cyclic light further suggest that this enzyme may be transferred from the apical processes to the ROS tips prior to shedding.

5'-Nucleotidase↗
PubMed1989

Identification and characteristics of a novel mitochondrial 5'-nucleotidase in rat liver.

In rat liver mitochondria there exists an AMP-dephosphorylating activity which converts external 5'-AMP to adenosine. It exhibits a pH optimum of 7.5 and a Km(AMP) of 0.085 mM. Furthermore, this activity is stimulated by magnesium (Km = 0.5 mM) and seems to be not affected by low concentrations of ATP or ADP. From the characteristics of the enzyme the existence of a 5'-nucleotidase in rat liver mitochondria which is localized on the outer surface of the inner mitochondrial membrane was concluded. The enzyme may be important for the production of cellular adenosine.

5'-Nucleotidase↗
PubMed1989

Antibodies to 5'-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate.

Human peripheral blood T cells were stimulated to proliferate when cultured with submitogenic doses of PMA and goat antibodies to 5'-nucleotidase (5'-NT). The degree of proliferation, as measured by [3H]TdR incorporation on day 3, was similar to that achieved by stimulation with PHA. Anti-5'-NT antibodies had no effect on PHA-induced proliferation. Maximal stimulation was achieved with 0.6 to 1.0 ng/ml of PMA and 125 micrograms/ml of IgG isolated from a goat anti-5'-NT antiserum. Both intact IgG and F(ab')2 fragments were stimulatory. IL-2R expression and IL-2 secretion were also induced by anti-5'-NT antibodies and PMA. Anti-5'-NT-induced proliferation was inhibited greater than 95% by a murine anti-IL-2 receptor mAb and required less than 0.3% monocytes. Similar results have been obtained with a murine mAb specific for 5'-NT. As expected, anti-5'-NT antibodies and PMA did not induce the proliferation of ecto-5'-NT-T cells isolated by cell sorting. Pretreatment of total T cells with phosphatidylinositol-specific phospholipase C removed an average of 89% of the 5'-NT activity from the cell surface and also inhibited by 83% the ability of the cells to proliferate in response to anti-5'-NT antibodies and PMA. Thus, the activation signal provided by anti-5'-NT antibodies is apparently transduced, in large part, by a form of the enzyme that is attached to the membrane via glycosyl-phosphatidylinositol linkage. These data suggest that 5'-NT may play a role in lymphocyte activation as has been proposed for other glycosyl-phosphatidylinositol-anchored lymphocyte surface proteins.

5'-Nucleotidase↗
PubMed1989

Observations on gamma-glutamyl transferase, 5'-nucleotidase and leucine aminopeptidase activities in the plasma of the horse.

In 18 horses there was no effect of age or sex on plasma activities of gamma-glutamyl transferase (gamma-GT), 5'-nucleotidase (5'-NT) and leucine aminopeptidase (LAP). All the enzymes were equally stable after storage for one month at -20 degrees C and there was no significant difference between their activities in serum and plasma in clinically normal horses. The pattern of release of gamma-GT, 5'-NT and LAP into plasma was studied in 114 horses which had a variety of orthopaedic, gastrointestinal, cardiovascular and hepatic (necrosis, lipidosis, neoplasia and cirrhosis) conditions. A definitive diagnosis of hepatic disease was established by histological examination of the liver. gamma-GT and 5'-NT were leaked into plasma in hepatic disease and gamma-GT was the more sensitive indicator of liver damage. There was some evidence that gamma-GT and 5'-NT plasma activities may increase in hepatic necrosis as well as in biliary obstruction. LAP was insensitive and not hepatic specific in the horse.

5'-Nucleotidase↗
PubMedDate not supplied

[Adenosine deaminase, purine nucleoside phosphorylase and ecto-5'-nucleotidase--markers of acute leukemia variants].

Estimation of enzymes participating in degradation of purine nucleotides--adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), ecto-5'-nucleotidase as well as the ratio of ADA/PNP in leukocytes was shown to be of importance in differential diagnosis of acute lymphoblast leukosis subforms and for identification of a nature of malignized leukocytes clone in acute undifferentiated leukosis. Importance of these analyses is determined by specific differences in distribution of the enzymatic activity in mononuclear cells in T-, non-T-, non-B-cell acute lymphoblast leukosis and acute myeloblast leukosis as well as due to similar level and ratios of these enzymatic activities in most cases of acute undifferentiated leukosis and in acute lymphoblast leukosis.

5'-Nucleotidase↗
PubMed1987

One antigen, one gold? A quantitative analysis of immunogold labeling of plasma membrane 5'-nucleotidase in frozen thin sections.

We present an evaluation of the efficiency of immunogold labeling for a low abundance plasma membrane protein. Several independent methods were used to determine the density of 5'-nucleotidase on the plasma membrane of the Fao cell. These methods include morphometry in combination with either enzymology or cell surface radiometric assay. Immunocytochemistry of frozen thin sections with either single or double layers of antibody and visualized with protein A complexed with 5 nm colloidal gold was used to estimate the same density. The application of a balance sheet to immunogold labeling demonstrates that the labeling is never quantitative. For example, labeling of the cell surface is always greater than labeling on the section. We show that departures from the "one antigen, one gold" ideal are systematic, so that an efficiency can be calculated and quantitative results can be obtained. The ability to obtain reliable quantitative results from immunogold labeling extends the utility of this already powerful technique.

5'-Nucleotidase↗
PubMed1987

Distribution of ecto-5'-nucleotidase on subsets of human T and B lymphocytes as detected by indirect immunofluorescence using goat antibodies.

Human T and B lymphocyte subsets were characterized for ecto-5'-nucleotidase (ecto-5'-NT) expression by two-color immunofluorescence by using polyclonal goat antibodies to 5'-NT and murine monoclonal antibodies to T and B cell subsets. Anti-5'-NT antibodies were prepared by immunizing a goat with purified human placental 5'-NT. Lymphocyte surface 5'-NT was detected with F(ab')2 fragments of immune goat IgG followed by biotinylated F(ab')2 rabbit anti-goat IgG and fluorescein isothiocyanate-avidin. Lymphocyte cell surface antigens were detected with phycoerythrin (PE)-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD16, and anti-CD19. HB-4, an antigen present on a major subset of human peripheral blood B cells, was detected with murine monoclonal anti-HB-4 and PE-anti-mouse-kappa. Analysis showed that ecto-5'-NT was expressed on 32 +/- 7% of CD3+, 19 +/- 6% of CD4+, and 50 +/- 21% of CD8+ T cells, but not on CD16+ lymphocytes. Ecto-5'-NT was also expressed on 81 +/- 8% of adult peripheral blood B cells as defined by PE-anti-CD19; HB-4 was expressed on 84 +/- 7% of CD19+ cells. The two populations of B cells were not identical, however, because HB-4 was co-expressed on only 79 +/- 18% of ecto-5'-NT+ B cells. Two-color immunofluorescent staining of T cells from a patient with congenital agammaglobulinemia and low T cell ecto-5'-NT activity revealed reduced percentages of ecto-5'-NT+ cells in his CD3+, CD4+, and CD8+ populations. Thus, reduced ecto-5'-NT activity by enzyme assay was paralleled by reduced numbers of 5'-NT molecules on the cell surface. Two-color immunofluorescent staining of B cells from a patient with hypogammaglobulinemia and low B cell ecto-5'-NT activity also revealed markedly reduced expression of 5'-NT. HB-4 expression was normal, however, suggesting that the patient's B cells were blocked in maturation subsequent to the acquisition of HB-4 but prior to that of ecto-5'-NT. These results demonstrate that anti-5'-NT antibodies will be valuable tools for analyzing ecto-5'-NT expression and lymphocyte maturation in patients with immuno-deficiency diseases.

5'-Nucleotidase↗