5-Nucleotidase activity in lymphocytes

Summary Some characteristics of lymphocyte 5-nucleotidase are reviewed. The optimal conditions for the cytochemical localization of 5-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that cytochemistry can be performed under conditions that do not lead to loss of AMPase activity. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. The splenic cell population included a distinct population of lymphocytes with readily demonstrable AMPase activity and another population with no cytochemically demonstrable AMPase activity. The number of positive cells varied, but was usually between 20 and 30% when cells were fixed in glutaraldehyde. Lymphocytes purified from thymus were always negative for cytochemically demonstrable AMPase activity. Biochemically, it was shown that the AMPase activity of spleen lymphocytes is more than six times higher than that of thymus lymphocytes. 5-Nucleotidase activity in normal and abnormal lymphocytes is discussed in the light of the latest findings. The possible function of 5-nucleotidase in lymphocytes is discussed.     

Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum.

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.     

Electron microscopic cytochemical characterization of bile canaliculi and bile ducts in vitro.

Electron microscopic cytochemical localization of Mg++-activated adenosine triphosphatase (Mg++-ATPase) and 5-nucleotidase (AMPase) was investigated in bile canaliculus-rich and bile duct-containing fractions isolated from rat liver. Comparative cyochemical studies between prefixed and non-prefixed fractions revealed that the activity of both enzymes could be detected in the fractions under appropriate experimental conditions. However, the cytochemical activity of AMPase was much more sensitive to glutaraldehyde than that of Mg++-ATPase. Mg++-ATPase and AMPase reaction products were localized primarily on bile canalicular microvilli, that is, along the outer (luminal) surface of canalicular plasma membranes, but they were never observed on bile ductal microvilli. AMPase was also detectable on lateral hepatic plasma membranes. Mg++-ATPase demonstrated by the cytochemical technique described is a reliable enzyme marker for isolated bile canalicular membranes. At high magnification, Mg++-ATPase reaction product was also observed on the microfilaments surrounding isolated bile canaliculi. The possibility that the reaction product on the pericanalicular microfilaments may result from the hydrolysis of ATP byan actomyosin ATPase-like enzyme associated with these filaments is briefly discussed.     

Macromolecular insoluble cold globulin (MICG): a novel protein from mouse lymphocytes. IV. Evidence for the plasma membrane distribution of MICG.

Macromolecular insoluble cold globulin is a glycoprotein synthesized predominantly by T lymphocytes in the mouse. The present report details experiments demonstrating the plasma membrane distribution of MICG on T lymphocytes. By utilizing immunofluorescent techniques it was shown that MICG was located in the external cell surface of 98% of thymic lymphocytes and 60% of splenic lymphocytes. Furthermore, in spleen cells, it was demonstrated that T cells and not B cells were surface MICG positive. Antibody to MICG was able to cap all of the immunofluorescent-positive (60%) spleen cells. In contrast, anti-MICG antibody did not induce cap formation on thymus cells. Only when dilute solutions of antibody were used did MICG cap on the thymus cells. Employing limited proteolysis of thymus and spleen cells MICG was shown to be regenerated on the surface of T cells with a half-life of 3.5 hr. The distribution and cell surface characteristics of MICG are discussed in terms of a "receptor-like" function for this protein.     

Ultracytochemical detection of nucleoside phosphatase activity in human peripheral blood cells

The authors elaborated and described the optimum conditions for fixation, incubation and preparation of human blood cell samples in minimum quantities for ultrastructural and ultracytochemical investigations of 5'-nucleotidase and ATPase activities. The best preservation of the blood cell ultrastructure was obtained after fixation with buffered 1% glutaraldehyde solution followed by postfixation in buffered 1% OsO4 solution. The best ultracytochemical demonstration of 5'-nucleotidase and ATPase activities was achieved after fixation in buffered 2% formaldehyde prior to cytochemical incubation. DMSO added to either fixation or incubation media was shown to damage the plasmalemma and glycocalyx structure in cell suspensions. ATPase in 5'-nucleotidase activities were revealed in plasmalemma, cytoplasmic reticulum, Golgi complex, mitochondria and in the nuclei, in particular, in the perinuclear space, nucleolus and chromatin. With respect to the localization and activity of nucleosidephosphatases, lymphocytes proved to be most heterogenic, with the enzyme activity level directly depending on the rate of ultrastructural differentiation in lymphocytes.     

The extracellular matrix proteins laminin and fibronectin modify the AMPase activity of 5'-nucleotidase from chicken gizzard smooth muscle

Laminin and fibronectin, but not collagen, affect the AMPase activity of the purified transmembrane protein 5'-nucleotidase. Laminin stimulates whereas fibronectin inhibits the AMPase activity of this ectoenzyme. The AMPase-modulating effects by these components of the extracellular matrix require a preincubation period of several hours when detergent-solubilized 5'-nucleotidase is employed, they can, however, instantaneously be elicited with liposome-incorporated 5'-nucleotidase.     

Vanadate inhibits degradation of short-lived, but not of long-lived, proteins in L-132 human cells.

We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms.     

Biochemical and enzymatic characterization of thymic and splenic lymphocyte plasma membranes from inbred rats.

Purified splenic and thymic lymphocytes from the ACI and F344 strains of inbred rats were disrupted by controlled hypotonic treatment, and their plasma membranes were prepared by sucrose density gradient centrifugation. The plasma membrane preparations were highly purified as judged by the structural appearance of the smooth membrane vesicles, by the 10- to 15-fold enrichment of 5'-nucleotidase, which cytochemically localized exclusively in the plasma membranes of intact lymphocytes, by the high cholesterol to phospholipid molar ratio (0.7-1.0), and by the very low specific activities of the enzymes associated predominantly with mitochondria, lysosomes, and endoplasmic reticulum. The protein and the lipid contents of the membranes were 48-55 and 37-48%, respectively. The total lipid content of plasma membranes was characteristically higher in thymic than splenic lymphocytes from both ACI and F344 strains. The specific activity of 5'-nucleotidase was similar in splenic lymphocyte membranes of the ACI strain, and in both the thymic and splenic lymphocyte membranes of the F344 strain. In contrast, the thymic lymphocyte membranes in the ACI strain showed half as much 5'-nucleotidase specific activity. Cytochemical results indicated that the 5'-nucleotidase is located on the outside surface of the lymphocyte plasma membranes.     

Reconstitution of purified chicken gizzard 5'-nucleotidase in phospholipid vesicles. Evidence for its transmembraneous character and the existence of functional domains on both sides of the phospholipid bilayer

5'-Nucleotidase, purified to homogeneity from chicken gizzard using published procedures [Dieckhoff, J., Knebel, H., Heidemann, M. and Mannherz, H. G. (1985) Eur. J. Biochem. 151, 377-383] was incorporated into artificial phospholipid vesicles after prolonged dialysis against detergent-free buffer or by a gel filtration procedure. After dialysis the obtained liposomes exhibit a mean diameter of 80 nm and contain 5'-nucleotidase at random orientation, demonstrated by finding up to 50% of the total liposome-incorporated AMPase activity to be cryptic, i.e. could only be measured after their permeabilization by addition of detergent. By affinity chromatography a phospholipid vesicle fraction could be obtained containing almost exclusively cryptic AMPase activity, thus representing the inside-out orientation of 5'-nucleotidase. Comparative analysis of physiochemical and enzymatic properties of 5'-nucleotidase reveals differences between the detergent-solubilized and the liposome-incorporated 5'-nucleotidase including a changed accessibility of the enzyme to polyclonal and monoclonal antibodies. Binding and AMPase inhibition studies with different polyclonal antibodies strongly indicate to the existence of a cytoplasmic domain of chicken gizzard 5'-nucleotidase. F-actin appears preferentially to interact with the cytoplasmic domain of liposome-incorporated 5'-nucleotidase.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

The redistribution of membrane surface immunoglobulin induces the rearrangement of some membrane integral proteins

We investigated whether the redistribution of surface membrane receptors is associated with rearrangement of integral membrane proteins. Using a newly developed process, which combines histochemical analysis with an immunofluorescence or immuno-electron microscopy-staining technique, we studied the redistribution of two membrane-bound enzymes, 5'-nucleotidase and ATPase, on mouse splenic lymphocytes and B lymphoma cells induced by anti-mouse immunoglobulin antibodies. Labeling and capping of the membrane surface immunoglobulin induced a similar rearrangement of both 5'-nucleotidase and ATPase from uniform distribution at 4 degrees C into 'patches' and caps at 37 degrees C.     

Comparison of 5'-nucleotidase activities among cultured murine melanoma cell lines

The activity of 5'-nucleotidase and ouabain-sensitive Na/K ATPase was determined in seven different mouse melanoma cell lines. Ouabain-sensitive Na/K ATPase activity was found in NP40-treated cell homogenates of all cell lines. However, 5'-nucleotidase activity was found in only one mouse melanoma cell line--JB/RH. The absence of expression of 5'-nucleotidase activity in the other six cell lines is not associated with pigmentation in melanoma cells, nor is the gene switched off in all transformed melanocytes of C57BL/6 origin.     

A histochemical study of variations in the localization of 5′-nucleotidase activity in the acinar cell of the rat exocrine pancreas over the twenty-four hour period

The circadian variation of 5'-nucleotidase (AMPase) activity was studied in rat pancreatic exocrine cells. The localization of this enzyme, often associated with the plasmalemma, was studied by ultracytochemical methods at six time points over the 24-h period. The localization of AMPase activity exhibited a clear-cut circadian variation. During the light span strong activity was observed on the luminal plasmalemma, negative or weak activity on the baso-lateral plasmalemma and clearly visible activity on intracellular structures such as cytoplasmic vacuoles (fragmentation-like vesicles), dilated rims of the Golgi cisternae (or cisternal ends of the Golgi stacks), condensing vacuoles and lysosomal bodies. During the dark span the activity was detectable only on the baso-lateral plasmalemma. The fact that AMPase activity could not be found on the luminal plasmalemma during the dark span suggests that the luminal membranes may be replaced by the membranes of secretory granules, which do not display AMPase activity. The intracellular localization of AMPase activity during the light span, especially at 08.00 h, includes all cytoplasmic compartments which have hitherto been associated with the intracellular pathway for membrane retrieval from the plasmalemma. Moreover, the appearance of the activity in the dilated rims of the Golgi stack and condensing vacuoles indicates that these compartments may constitute a functional unit.     

Lymphocyte ecto-5'-nucleotidase activity in infancy: increasing activity in peripheral blood B cells precedes their ability to synthesize IgG in vitro

Ecto-5'-nucleotidase activity was measured in peripheral blood lymphocytes isolated from serial specimens from nine healthy full-term infants and two premature infants at 0, 2, 4, and 6 mo of age. The postnatal nadir in activity was 7.1 +/- 2.0 nmol/hr/10(6) cells, which is the same as the activity in cord blood lymphocytes (7.0 +/- 2 nmol/hr/10(6) cells). The activity rose twofold to 13.2 +/- 3.8 nmol/hr/10(6) cells at 6 mo of age (p less than 0.001, paired t-test), which is similar to the activity in adult peripheral blood lymphocytes (14.1 +/- 6.3 nmol/hr/10(6) cells). This increased activity in total lymphocytes reflects increased activity in the B cell population. B cell ecto-5'-nucleotidase activity in two infants at 12 to 13 mo of age was 19.3 and 25.2 nmol/hr/10(6) cells, values that are four-to fivefold higher than for cord blood B cells (5.6 +/- 2.8 nmol/hr/10(6) cells) and within the normal range for adult B cells (27.9 +/- 12 nmol/hr/10(6) cells). In spite of a greatly expanded peripheral blood B cell population, studies of immunoglobulin biosynthesis in vitro demonstrated that infant peripheral blood B cells are functionally immature with no synthesis of IgG in response to Epstein Barr virus. Thus, the increase in peripheral blood B lymphocyte ecto-5'-nucleotidase activity in infants precedes their acquisition of a capacity for IgG synthesis in vitro. Data from a hypogammaglobulinemic infant revealed a persistently low ecto-5'-nucleotidase activity over a 10-mo period until at 14 mo of age the activity was 8.8 nmol/hr/10(6) cells in total lymphocytes and 13.0 nmol/hr/10(6) cells in B cells, which correlated with in vivo and in vitro evidence of delayed B cell maturation. Thus, ecto-5'-nucleotidase activity may be a useful cell surface marker in studies of human postnatal B cell maturation.     

3'-nucleotidase activity in procyclic and bloodstream stages of Trypanosoma rhodesiense

Both procyclic and bloodstream-derived trypanosomes of Trypanosoma rhodesiense exhibit a nucleotidase activity which is capable of hydrolyzing 3'-ribonucleotides. Nucleotidase activity in assays employing 5'-nucleotide substrates is not detectable in preparations of either phenotype. The 3'-nucleotidase activity in lysates of procyclic trypanosomes is pelletable at 100,000 g, whereas that activity from bloodstream forms is operationally soluble under the conditions employed. The 3'-nucleotidase activities of both stages share the following characteristics: pH optimum of 8.5, substrate preference for purine ribonucleotides, resistance to commonly used phosphatase inhibitors, including fluoride, tartrate and molybdate ions. Both activities are reversibly inhibited by EDTA and reactivated by Co2+ ions. As visualized by fine-structure cytochemical methods, 3'-nucleotidase activity was distributed over the entire surface of procyclic trypanosomes, but not bloodstream parasites bearing an intact surface coat.     

Location of nucleotide pyrophosphatase and alkaline phosphodiesterase activities on the lymphocyte surface membrane.

1. Isolated mouse spleen lymphocytes hydrolysed UDP-galactose added to the medium. Nucleotide pyrophosphatase activity that accounted for this hydrolysis was enriched to a similar extent as alkaline phosphodiesterase and 5'-nucleotidase in a lymphocyte plasma-membrane fraction. 2. The cell surfaces of mouse spleen and thymus lymphocytes were iodinated with 125I by using the lactoperoxidase-catalysis method. Detergent extracts of the cells were mixed with a purified anti-(mouse liver plasma-membrane nucleotide pyrophosphatase) antiserum and the immunoprecipitates analysed by polyacrylamide-gel electrophoresis. Only one major radioactive component, similar in size (apparent mol.wt 110000-130000) to the liver enzyme, was observed. 3. Electrophoresis of an iodinated spleen plasma-membrane fraction indicated peaks of radioactivity, including one of apparent mol.wt 110000-130000. 4. When detergent extracts of spleen lymphocytes were passed through a Sepharose-bead column containing covalently attached anti-(nucleotide pyrophosphatase) antiserum, the nucleotide pyrophosphatase activity was retained by the beads, whereas protein and leucine naphthylamidase activity were eluted. 5. The results indicate that nucleotide pyrophosphatase and alkaline phosphodiesterase activities are due to the location of the same or similar enzymes at the outer aspect of the lymphocyte plasma membrane. Some possible functions of enzymes at this location are discussed.     

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.     

Affinity labelling of 5''-nucleotidases with 5''-p-fluorosulphonylbenzoyladenosine.

5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'-nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.     

Ecto-5'-nucleotidase activity in lymphoblastoid cell lines derived from heterozygotes for congenital X-linked agammaglobulinemia

Lymphoblastoid cell lines were established from female relatives of patients with congenital X-linked-agammaglobulinemia by Epstein-Barr virus transformation of their peripheral B lymphocytes. Cell lines derived from presumed carriers were characterized by low ecto-5'-nucleotidase activity and a reduced percentage of surface immunoglobulin-bearing cells. Measurement of ecto-5'-nucleotidase activity in newly established lymphoblastoid cell lines may provide a means for the identification of heterozygotes for congenital X-linked agammaglobulinemia.     

Implications of a 5'-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5'-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56 degrees C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37 degrees C for 1 h and it was destroyed completely by heating at 100 degrees C for 30 min. When the heated (56 degrees C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of N-acetyl-beta-D-glucosaminidase or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5'-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35,000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggest that the previously reported undetectability of 5'-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5'-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5'-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.