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PubMed1988

5'-Nucleotidase and adenosine deaminase in developing fetal guinea pig brain and the effect of maternal hypoxia.

The activity of key enzymes of adenosine metabolism was studied in the developing fetal guinea pig brain. The activities of 5'-nucleotidase and adenosine deaminase were determined in the brains of fetal guinea pigs at 30, 35, 40, 45, 50, 55, and 60 days of gestation. The level of 5'-nucleotidase activity was extremely low at 30 and 35 days of gestation but increased rapidly during the 40 to 60 day period. The enzyme activity increased in the presence of Mg2+ with the Mg2+ - dependent activation increasing with the age of gestation. This Mg2+ - dependent activity was primarily associated with the membrane fraction. Prenatal hypoxia significantly increased the fetal brain M2+ - independent 5'-nucleotidase activity at 45 days of gestational age and beyond. Prior to this age, no effect was evident. Furthermore, following hypoxia, the Mg2+ - dependent activation of 5'-nucleotidase activity was lost. The activity of adenosine deaminase was present at 30 days of gestation and, unlike 5'-nucleotidase, it remained at the same level until 60 days. The results indicate that the term fetal guinea pig brain has the enzymatic mechanisms of adenosine metabolism and thus the potential for adenosine-mediated regulation of cerebrovasculature during hypoxia.

5'-Nucleotidase↗
PubMed1983

A family of phosphohydrolases from bovine intestinal mucosa: 5'-nucleotidase.

Bovine intestinal 5'-nucleotidase has been partially purified and characterized for comparison with two other phosphohydrolases from the same tissue, alkaline phosphatase and 5'-nucleotide phosphodiesterase, which are closely related structurally and mechanistically. Kinetic studies with a variety of nucleotides and phosphonate analogs show that, although 5'-nucleotidase is a monoesterase like alkaline phosphatase, it more closely resembles 5'-nucleotide phosphodiesterase in its high affinity and specificity for nucleotide binding. 5'-Nucleotidase is bound very strongly by an affinity column containing a bound phosphonate analog of ADP but is not bound by an affinity column containing a non nucleotide phosphonate which selectively binds alkaline phosphatase. 5'-Nucleotidase is strongly bound by immobilized antibodies prepared against 5'-nucleotide phosphodiesterase, and is less strongly bound by immobilized antibodies prepared against alkaline phosphatase. We conclude that 5'-nucleotidase is structurally more similar to 5'-nucleotide phosphodiesterase than to another monoesterase, alkaline phosphatase.

5'-Nucleotidase↗
PubMed1982

Absence of 5'-nucleotidase in porcine polymorphonuclear leucocyte membranes.

A fraction enriched in plasma membranes from porcine polymorphonuclear leucocytes, isolated by sucrose density centrifugation was shown to possess considerable AMP hydrolysing activity (150 nmol/min per mg protein). However all of this activity could be inhibited using excess p-nitrophenyl phosphate in the incubation medium. Furthermore the hydrolysis of AMP by the membrane was unaffected by the 5'-nucleotidase inhibitor alpha, beta-methyleneadenosine diphosphate and by the lectin concanavalin A, another potent inhibitor of 5'-nucleotidase. An antibody against mouse liver 5'-nucleotidase also did not inhibit the activity. These results suggest that the hydrolysis of AMP by porcine polymorph membranes is not accomplished by a specific 5'-nucleotidase and the necessity for distinguishing between true 5'-nucleotidase and non-specific phosphatase activity is discussed.

5'-Nucleotidase↗
PubMed1982

Properties of lymphocyte 5'nucleotidase in normal subjects and patients with chronic lymphocytic leukemia.

Heterogeneity for 5'nucleotidase has been demonstrated in chronic lymphocytic leukemia [12]. The enzyme is not detectable in the lymphocytes from the majority of patients with this disorder, but normal and even supranormal levels are found in some cases [17]. In the present studies, the properties of this ecto-nucleotidase were investigated in unhomogenized lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia. The activity was found to have a pH optimum of 8.0-8.5 and a preference of 5'ribo- over 5'deoxyribonucleotides. The enzyme was inactive towards 2',3'AMP. The Km for AMP showed a broad range from 19 to 210 microM in unhomogenized lymphocytes. An unexpected relationship was observed between sp. act. and this Km in that higher Km values were noted with cells from subjects with high lymphocyte 5'nucleotidase sp. act. and low Km values in lymphocyte preparations with low sp. act. When plasma membranes were prepared, a narrow range of low Km values unrelated to sp. act. was noted. The change in Km was not due to Pi concentration or nucleotide effects. The ecto-enzyme was inhibited by alpha, beta-methylene adenosine diphosphate, while cytosol phosphatase activity was not inhibited by this compound. Heat stability studies revealed a 45% loss in 5'nucleotidase activity after incubation for 20 min at 65 degrees C. A comparison of the substrate preference, Km values, effect of inhibitor and subcellular localization revealed no differences between the enzyme from patients with chronic lymphocytic leukemia and from normal subjects. This finding is consistent with the suggestion that the undetectable or supranormal levels observed in the lymphocytes of patients with this disorder stems from variations in the percentage of cells having 5'nucleotidase rather than changes in the enzyme proper.

5'-Nucleotidase↗
PubMed1983

Regional effects of hypothyroidism on 5'-nucleotidase and cyclic nucleotide phosphohydrolase activities in developing rat brain.

The activities of 5'-nucleotidase (EC 3.1.3.5) and of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) (EC 3.1.4.37) were measured in crude membrane preparations from 6 brain regions of hypothyroid rats 31 days and 42 days after conception (8-9 days and 19-20 days after birth respectively) and from normal rats 31, 36 and 42 days after conception. At 42 days the activities of both enzymes were markedly and significantly lower than in controls in all hypothyroid brain regions. The pattern of increase in CNP activity in the brain regions of normal animals reflected the caudo-rostral progress of myelination. At 42 days postconception in the more caudal regions of hypothyroid brain, myelination, as assessed by CNP activity, was delayed by 5-7 days. In the more rostral regions where normal activity had not yet developed, the delay due to hypothyroidism was not clearly defined. The lowered activity of 5'-nucleotidase observed using crude membrane preparations from hypothyroid animals was shown not to be due to a redistribution of enzyme between cytosol and membranes or to losses into the supernatant fraction during preparation of the particulate fraction used for assays. In addition the lowered activity of 5'-nucleotidase could not be accounted for in terms of the delayed myelination and consequent absence of myelin-associated enzyme. In contrast to the results with CNP, the effects of hypothyroidism on 5'-nucleotidase in the 6 regions showed no caudo-rostral pattern. Although it was clear that the lowered activity of 5'-nucleotidase in hypothyroid brain at 20 postnatal days was not directly attributable to the state of delayed myelination, no identification of the structures or cell types affected could be made.

2',3'-Cyclic Nucleotide 3'-Phosphodiesterase↗
PubMed1988

5'-Nucleotidase in postnatal ontogeny of rat cerebellum: a marker for migrating nerve cells?

The distribution of the ectoenzyme 5'-nucleotidase was studied in the developing rat cerebellum using enzyme- and immunocytochemical techniques in order to evaluate its appearance in the processes of cellular migration during nervous system development. 5'-Nucleotidase enzymatic activity appeared first in the external granular layer (postnatal day 6). Then, the inner portion of the external granular layer and the developing molecular layer became prominently stained (postnatal day 12). At 21 days the adult pattern was established with a diffuse labeling of the whole molecular layer. Additionally, 5'-nucleotidase enzymatic activity was found around Purkinje cells. At the ultrastructural level migrating nerve cells exhibited an intense surface labeling whereas resting neuroblasts of the external granular layer as well as all post-migratory nerve cells were negative. Immunocytochemistry differed from enzyme histochemistry in that immunoreactivity was found to be selectively associated with glial elements. It is suggested that 5'-nucleotidase may serve as a marker for processes of neuronal migration and neuron-glia interactions during postnatal ontogeny. Underlying mechanisms could be due to an interaction of 5'-nucleotidase with the extracellular matrix molecule, laminin, and cytoskeletal actin involved in locomotion.

5'-Nucleotidase↗
PubMed1983

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.

5'-Nucleotidase↗
PubMed1984

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact.

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.

5'-Nucleotidase↗
PubMed1985

5'-Nucleotidase activity of isolated mature rat cardiac myocytes.

Specific location of 5'-nucleotidase in the heart has been uncertain, some authors citing evidence for an exclusively non-myocyte location, while other data point to the existence of cytoplasmic and membrane-bound fractions. Single myocytes isolated from mature rat heart, and free of endothelial or interstitial cells, have been used to establish that muscle cells of the myocardium are rich in 5'-nucleotidase, exhibiting activity sufficient to account for the total myocardial content of this enzyme. All 5'-nucleotidase is accessible to extracellular AMP. Inhibitors of 5'-nucleotidase and adenosine transport have been used to establish that only the adenosine component of adenine nucleotides is taken up by myocytes, but hydrolysis of AMP by 5'-nucleotidase does not commit the adenosine formed to transport across the sarcolemmal membrane. Myocytes also have ecto-phosphatases which hydrolyse ADP and ATP.

5'-Nucleotidase↗
PubMed1986

Demonstration of the association of the cell-surface enzyme, 5'-nucleotidase, with microvillar microfilaments by phalloidin shift on velocity sedimentation gradients.

The cell-surface enzyme 5'-nucleotidase in microvilli from 13762 rat mammary adenocarcinoma cells remains largely associated with microfilament-containing high-speed pellets from Triton X-100 extracts of the microvilli. The fraction remaining with the insoluble portion is higher under ionic conditions which enhance microfilament stability. To minimize trapping and cosedimentation we have analyzed the distribution of microfilaments and 5'-nucleotidase activity on velocity sedimentation sucrose gradients of the microvillar extracts. A large fraction of the total enzyme activity is found in the filament fractions in the middle of the gradient. When phalloidin is included in the extraction buffer to stabilize the microfilaments, both the microfilaments and the bulk of the nucleotidase activity are shifted further into the gradients. Both the position of the filament fraction and the percentage of the total nucleotidase activity remaining with the filament fraction varies with extraction buffer composition and conditions. Nonetheless, under all conditions tested, a large percentage of the activity was shifted, along with the microfilaments, in the presence of phalloidin. These results are consistent with a specific association of 5'-nucleotidase with microfilaments in the ascites tumor cell microvilli.

5'-Nucleotidase↗
PubMed1984

Interactions with lectins indicate differences in the carbohydrate composition of the membrane-bound enzymes acetylcholinesterase and 5'-nucleotidase in different cell types.

We have examined the interactions of the membrane-bound enzymes, 5'-nucleotidase and acetylcholinesterase from bovine tissues with lectins and shown that glycosylation contributes significantly to the polymorphism of these enzymes, in a tissue-specific manner. Lectins which bind 5'-nucleotidase also inhibit its catalytic activity to various degrees. We found different specificities with 5'-nucleotidases from various cell types: for example lymphocyte 5'-nucleotidase did not interact with wheat germ agglutinin, in contrast with 5'-nucleotidases from hepatocyte and caudate nucleus membranes. Treatment with glycohydrolases, alpha-D-mannosidase and neuraminidase, suggested that the latter enzymes possess sialic residues which are absent in the lymphocyte enzyme. Interactions of acetylcholinesterase with lectins were demonstrated by sedimentation analysis and binding to immobilized lectins, but its activity was generally not affected. A notable exception was lymphocyte acetylcholinesterase which was inhibited by the fucose-binding Ulex europeus agglutinin. This inhibition was relieved by alpha-L-fucose but not by alpha-D-fucose and reduced after treatment with alpha-L-fucosidase. In addition this enzyme differs from acetylcholinesterases from other tissues by its higher Km value, although it appears immunologically equivalent. The different forms of acetylcholinesterase from the same tissue may differ in their interactions with lectins. In muscle for example G4 carries carbohydrate chains of the complex type whereas G1 appears to possess only the high mannose type. We discuss the possible relationships between these forms.

5'-Nucleotidase↗
PubMed2005

The 5'-nucleotidases as regulators of nucleotide and drug metabolism.

The 5'-nucleotidases are a family of enzymes that catalyze the dephosphorylation of nucleoside monophosphates and regulate cellular nucleotide and nucleoside levels. While the nucleoside kinases responsible for the initial phosphorylation of salvaged nucleosides have been well studied, many of the catabolic nucleotidases have only recently been cloned and characterized. Aside from maintaining balanced ribo- and deoxyribonucleotide pools, substrate cycles that are formed with kinase and nucleotidase activities are also likely to regulate the activation of nucleoside analogues, a class of anticancer and antiviral agents that rely on the nucleoside kinases for phosphorylation to their active forms. Both clinical and in vitro studies suggest that an increase in nucleotidase activity can inhibit nucleoside analogue activation and result in drug resistance. The physiological role of the 5'-nucleotidases will be covered in this review, as will the evidence that these enzymes can mediate resistance to nucleoside analogues.

5'-Nucleotidase↗
PubMed1986

Subcellular distribution of cardiac 5'-nucleotidase: alteration of microsomal pool in hypertrophied pig heart.

The subcellular distribution of the 5'-nucleotidase activity was investigated in normal and hypertrophied pig hearts; normal rat hearts were used for comparison. The left ventricular hypertrophy was induced in pigs by banding the supravalvular aorta for 4, 8 and 12 weeks. By employing different procedures for the isolation of cardiac membranes, a major catalytic site for 5'-nucleotidase was found to be located at sarcolemma in rat heart and microsomes (sarcoplasmic reticulum) in pig heart. A progressive decrease in the homogenate and microsomal 5'-nucleotidase activity occurred upon the development of myocardial hypertrophy in pigs. This reduction in microsomal 5'-nucleotidase activity was characterized by a depression in both apparent Vmax and Km values. These results indicate that a primary 5'-nucleotidase pool is present in the intracellular compartment of the pig heart and is altered during the development of hypertrophy.

5'-Nucleotidase↗
PubMed1987

Is ecto-5'-nucleotidase essential for stimulation of human lymphocytes? Evidence against a role of the enzyme as mitogenic lectin receptor.

We studied with specific polyclonal and monoclonal antibodies against human ecto-5'-nucleotidase whether the enzyme, located on the surface of human peripheral lymphocytes, could function as a mitogenic receptor for the lectins PHA, Con A and PWM. Strong, but unspecific inhibitory effects on lymphocyte stimulation are observed with unfractionated antisera and ascitic fluids. However, when purified IgG from these sources is used, no such effects are found, while at the same time, complete inhibition of ecto-5'-nucleotidase activity is maintained. It is concluded that the enzyme does not act as a mitogenic receptor for the lectins. When purine de novo synthesis of the lymphocytes is blocked by aminopterin and purine nucleotides in the extracellular medium are given as the only purine source, lymphocyte stimulation becomes dependent on the enzymatic activity of ecto-5'-nucleotidase. This is independent of the lectin used. Under these conditions, enzyme activity on the 20-30% 5'-nucleotidase-positive cells is necessary and is sufficient to support the stimulation of the whole culture. In these cultures, anti-5'-nucleotidase-IgG completely depresses cell proliferation, showing clearly that this is the only enzyme on the lymphocyte surface that is capable of degrading extracellular nucleotides.

5'-Nucleotidase↗
PubMed1986

The membrane topography of ecto-5'-nucleotidase in rat hepatocytes.

The transmembrane topography of the rat hepatocyte ectoenzyme 5'-nucleotidase was studied by the use of glycoprotein labelling and limited-proteolysis techniques. Comparison, by one-dimensional peptide mapping, of enzyme iodinated from outside the cell with that iodinated in the solubilized state showed that no additional iodination sites were revealed on solubilization. Incubation of newly synthesized enzyme in a microsomal membrane fraction with proteinase showed that the entire molecule of 5'-nucleotidase was protected from proteolysis. These data suggest that little, if any, of the 5'-nucleotidase molecule is present on the cytoplasmic side of the plasma membrane. No evidence was found for a previously proposed interaction between 5'-nucleotidase and actin, although the ability of preparations of 5'-nucleotidase to prevent inhibition of deoxyribonuclease I by actin was explained by minute traces of ATPase activity. Comparison of peptide maps of enzyme labelled by iodination or by methods specific for carbohydrate showed that in both cases predominantly one section of the molecule was labelled. It is proposed that the enzyme is a short-stalked integral membrane protein without a cytoplasmic domain in which about one-third of the molecule forms the accessible molecular surface.

5'-Nucleotidase↗
PubMed1988

The pigeon heart 5'-nucleotidase responsible for ischaemia-induced adenosine formation.

1. A 5'-nucleotidase with a strong preference for AMP over IMP was characterized in homogenates and subcellular fractions of pigeon heart by using concentrations of ATP, ADP and AMP which mimicked those present in the ischaemic tissue. 2. The AMP-5'-nucleotidase had a neutral pH optimum and an apparent Km in the range 4.6-5.2 mM. It was stimulated by ATP plus ADP, and was inhibited by other nucleoside monophosphates, Pi and p-nitrophenyl phosphate, but not by ribose 5-phosphate or beta-glycerophosphate. The enzyme was not inhibited by [alpha beta-methylene] ADP or by 5'-deoxy-5'-isobutylthioadenosine, an inhibitor of the previously purified IMP-preferring cytosolic 5'-nucleotidase. 3. Subcellular-fractionation studies indicated that the enzyme has access to cytosolic AMP, although it may be associated by weak ionic interactions with an organelle present in the low-speed particulate fraction. 4. A 5'-nucleotidase was detected under similar conditions in homogenates of rat heart. 5. The activity of the pigeon heart AMP-5'-nucleotidase was sufficient to account for previously measured rates of ischaemia-induced adenosine formation. The similar activity in rat heart could, however, account for only part of ischaemia-induced adenosine formation in this tissue.

5'-Nucleotidase↗
PubMed1989

5'-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan.

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.

5'-Nucleotidase↗
PubMed1985

An improved procedure for purifying 5'-nucleotidase from various sources. Evidence for tissue and species differences in their molecular mass and affinity for F-actin.

5'-Nucleotidase from chicken gizzard smooth muscle has been extracted, using a sulfobetaine derivate of cholic acid, and purified to homogeneity by employing three chromatographic steps. It is shown that the purification scheme can be applied to 5'-nucleotidase from other sources, such as rat liver. On sodium dodecyl sulfate polyacrylamide gels, stained with silver nitrate, the purified enzyme from chicken gizzard shows a single polypeptide band with an apparent molecular mass of 79 kDa. The enzyme purified from rat liver exhibits a molecular mass of 73 kDa in agreement with published data [Bailyes, E.M., Soos, M., Jackson, P., Newby, A. C., Siddle, K. & Luzio, J.P. (1984) Biochem. J. 221, 369-377). Gel filtration, using non-denaturating detergent solutions, indicates that the native enzyme may exist as a homodimer (152 kDa) or homotetramer (310 kDa). Antibodies raised against the enzyme purified from chicken gizzard bind only 5'-nucleotidase, solubilized from chicken muscular sources, when immobilized, but not from chicken or rat liver. The existence of tissue specific variants of 5'-nucleotidase is therefore postulated and it appears that these particular isoforms can also be classified in membranous and secretory forms of 5'-nucleotidase. They also differ in their mode of interaction with actin. The AMPase activity of the membranous (= muscular) isoform is inhibited to a considerably higher percentage by F-actin than the enzyme isolated from rat liver.

5'-Nucleotidase↗