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PubMed1987

The distribution of adenosine A1 receptors and 5'-nucleotidase in the brain of some commonly used experimental animals.

The distribution of adenosine A1 receptors was studied quantitatively in the brain of the rat, mouse, guinea-pig and cat, using in vitro autoradiography with [3H]N6-cyclohexyladenosine as ligand. Preliminary binding studies in brain sections from the guinea-pig and cat gave results similar to previous data from the rat and showed that the binding site had the pharmacological profile of an A1 receptor. The overall distribution of receptors was comparable in the species studied. The receptors were concentrated in the hippocampus, the cerebral cortex, some thalamic nuclei, the basal ganglia and the cerebellar cortex. The hypothalamus and the brainstem were sparse in receptors. Differences among species in receptor distribution and/or density were seen in some regions, e.g. the cerebral cortex, the striatum, the lateral geniculate nucleus and the cerebellar cortex. The autoradiograms were compared with adjacent sections stained for 5'-nucleotidase. There was in general a poor correlation between the distribution of A1 receptors and 5'-nucleotidase. Furthermore, there were marked differences between species in the distribution of the enzyme. The species differences observed in receptor localization may be of functional relevance.

5'-Nucleotidase↗

PubMed1988

The role of 5'nucleotidase in therapy-resistance of childhood leukemia.

Purine nucleotides become available for a cell by two routes, namely purine de novo synthesis and the purine salvage pathway. 5'Nucleotidase is a purine pathway enzyme and is present in two forms. Cytoplasmic 5'nucleotidase (cyto 5'NT) catalyzes the intracellular degradation of purine nucleotides into their corresponding nucleosides. The plasma membrane bound form (ecto 5'NT) has its active site facing the external medium. Its function is the extracellular dephosphorylation of nucleotides, to which cells are generally impermeable, into nucleosides and the transport of these nucleosides through the cell membrane. Lymphoblastic 5'NT activity varies between different children with common-ALL. 5'NT positive cases have a higher relapse rate and thus a poorer prognosis than 5'NT negative cases. This can be explained by two hypotheses which are not mutually exclusive: 1. Rescue hypothesis. When purine de novo synthesis is blocked by methotrexate (MTX) and/or 6-mercaptopurine (6-MP), the malignant cell has to rely on the purine salvage pathway. This pathway depends on the ecto-5'NT activity. So, leukemic cells might be resistant to MTX and/or 6-MP because of ecto-5'NT activity. 2. Breakdown hypothesis. Leukemic cells are resistant to 6-MP because of the breakdown of the toxic nucleotide form of 6-MP into the nucleoside form by cyto-5'NT.

5'-Nucleotidase↗

PubMed1983

Effects of phenolic compounds on 5-nucleotidase activity in some tissues of Notopterus notopterus (Pallas): a biochemical study.

In the present study, effects of phenol (P), 2,4-dinitrophenol (DNP) and pentachlorophenol (PCP) individually, and in three combinations viz., (PCP + DNP)/P (highly antagonistic), (DNP + P)/PCP (additive) and (P + DNP)/PCP (highly synergistic) at three sublethal levels (1/10, 1/15, and 1/20 fractions of 96 h LC50) on 5-nucleotidase activity have been studied in brain, liver, kidney and gills of a freshwater teleost, Notopterus notopterus. The results indicate that 5-nucleotidase activity was inhibited significantly (P less than 0.05; P less than 0.01; P less than 0.001) in these tissues at most of the concentrations after 15 and 30 days of exposure. However, significant stimulation (P less than 0.05) of this enzyme was also notice in kidney at 1/20 of P, and (PCP + DNP)/P combination after 15 days of exposure. Inhibition of this enzyme in tissues possibly represents the uncoupling of oxidative phosphorylation, while stimulation of the enzyme indicates that the concerned phenolic compounds might be inducing the cells of the kidney to produce more of this enzyme.

2,4-Dinitrophenol↗

PubMed1988

5'-Nucleotidase in skin fibroblasts from patients with Duchenne muscular dystrophy.

The 5'-nucleotidase of plasma membranes of cultured skin fibroblasts from patients with Duchenne muscular dystrophy had a reduced affinity for its substrate, 5'-AMP. The Arrhenius plot of the temperature dependence of this enzyme activity was normal. There was no difference between patients and controls in the specific 5'-nucleotidase activity in the whole cell homogenates.

5'-Nucleotidase↗

PubMedDate not supplied

Effects of 4-hydroxynonenal on adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes.

Adenylate cyclase and 5'-nucleotidase activities in rat liver plasma membranes were assayed in vitro in the presence of 4-hydroxy-2,3-trans-nonenal (HNE), a major end-product of microsomal lipid peroxidation. Both basal and glucagon-stimulated adenylate cyclase were inhibited in a dose-dependent manner, even at micromolar HNE concentrations, whereas fluoride-stimulated activity increased. A biphasic, dose- and time-dependent effect was noted when the basal activity was monitored at increasing doses. 5'-Nucleotidase activity was also decreased by HNE, but only at millimolar concentrations. These findings are related to the view that aldehydes, especially HNE, may act as diffusible cytotoxic compounds when lipid peroxidative derangement of membrane lipids is provoked by toxic conditions.

5'-Nucleotidase↗

PubMed1980

Serum 5'-nucleotidase of a breast cancer patient.

Serum derived from the breast cancer patient included in this study was found to be a rich source of 5'-nucleotidase. In addition, it also contains nonspecific alkaline phosphatase. The properties of 5'-nucleotidase were studied by eliminating the interference of serum non-specific alkaline phosphatase by the preliminary incubation of serum in glycine-NaOH buffer containing ethylenediamine tetraacetate and magnesium. The enzyme has a pH optimum at 9.5. It is remarkably stable when held at 50 degrees C at pH 7.5, but it readily lost activity in the acid medium at pH 4.2. It hydrolyzes both the ribo- and deoxyribo-nucleoside 5'-phosphates. It has the highest preference for cytidine 5'-phosphate. Adenosine 2'- or 3'- phosphates are refractory to its action.

5'-Nucleotidase↗

PubMed1987

Biochemical evidence for Mn2+-dependent 5'-nucleotidase activity in isolated rod outer segments.

Recently cytochemical evidence has been presented for a novel enzyme activity, i.e. 'manganese-dependent pyrimidine 5'-nucleotidase (MDPNase)' activity in the rod outer segments (ROS) of rat retinas in situ and in isolated rat ROS. The present biochemical study was undertaken to seek further evidence for this enzyme activity using an independent method. A series of enzyme assays was carried out to test for MDPNase activity in Triton extracts of rat ROS isolated by sucrose density gradient centrifugation. Hydrolysis of the substrate, cytidine-5'-monophosphate, was measured spectrophotometrically and expressed as microgram of released inorganic phosphorus hr-1 mg-1 protein in the sample. The results showed that the ROS extracts contained enzyme activity (18.1 +/- 3.8) that was increased 5-6-fold (102.3 +/- 10.6) in the presence of 7.4 mM MnCl2. The enzyme activity was not enhanced by Mg2+ ions (19.0 +/- 7.7) and was strongly inhibited by 10-20 mM NaF (11.8 +/- 2.9). Assays for substrate specificity revealed that the Mn2+-stimulated phosphatase activity was specific for 5'-nucleotides. Pyrimidine nucleotides (5'-CMP and 5'-UMP) were the preferred substrates. Comparison of enzymatic hydrolysis of 5'-CMP and 5'-AMP over a pH range from 4.5 to 8.0 revealed that at acid pH, the majority of the observed 5'-nucleotidase activity (82% at pH 5.0, 58% at pH 5.5) was manganese dependent, whereas at neutral pH and above, most of the enzyme activity was unaffected by the presence of Mn2+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗

PubMed1988

Ultrastructural cytochemical localization of 5'-nucleotidase activity in axon-myelin-Schwann cell complex.

5'-Nucleotidase activity has been localized at the ultrastructural level in the axon-myelin-Schwann cell complex. Sciatic nerves of rabbits of pre- and postnatal development were used. Positive reaction was found on the plasma membrane, basal lamina, cytoplasm, and finger-like processes of the Schwann cells; on the intraperiod lines of the compact myelin, on the surface of myelin sheath, in the split myelin lamellae in the paranodal regions and Schmidt-Lanterman clefts, in segments of outermost and innermost lamellae, split off from the interparanodal myelin, in the mesaxons (outer and inner), in the loose myelin lamellae in the earlier stages of myelinization; on the axolemma (especially in the nodal and paranodal segments), in the periaxonal space, axoplasm. The alterations of 5'-nucleotidase distribution were associated with the developing myelin sheath.

5'-Nucleotidase↗

PubMed1989

Cerium-induced light-microscopic demonstration of 5'-nucleotidase activity in the lymphatic capillaries of the proximal oesophagus of the rat.

Lymphatic capillaries with narrow lumina cannot be identified with certainty by means of conventional light microscopy. This, however, can be achieved with great precision by means of labelling the 5'-nucleotidase activity which is high in lymphatic capillaries. In blood capillaries, on the other hand, it is either lacking or much less pronounced depending on the organ. The reactions performed thus far occurred primarily in the presence of lead ions which had a toxic effect on the enzymes. In cerium, however, this inhibiting influence can be neglected. 2 light-microscopic procedures of labelling the 5'-nucleotidase activity, which make use of cerium, are presented and compared here for the first time. The cerium perhydroxide technique is especially suited for demonstrating the lymphatic capillaries. In comparison to the previous enzyme histochemical methods, a definitely more precise demonstration of the endothelia of small lymphatics now is possible. The reason for this is that this method only reacts to regions of high enzyme activity. As opposed to previous tests on cryostat sections, the lumen of the lymphatic capillary is nearly always identifiable as such if employing this type of reaction. These properties of the cerium-perhydroxide reaction allow definite statements regarding the distribution of the lymphatic capillaries in the tissue. Its simple and rapid course of reaction favour the use of this method in clinical problems. In the other technique presented, the manganese dioxide technique, the demonstration of lymphatic capillaries is by far less exact due to its greater sensitivity even to low enzyme activities.

5'-Nucleotidase↗

PubMed1985

5'-Nucleotidase activity and substrate affinity in digenetic trematodes.

5'-nucleotidases of eight species of digenetic trematodes were studied using five different substrates. All species showed the following preferential order of substrate affinity; AMP greater than CMP greater than GMP greater than TMP greater than UMP. It was observed that different species occupying similar habitats possessed closely related levels of enzyme activities. The function of 5'-nucleotidases in trematodes is also suggested.

5'-Nucleotidase↗

PubMed1989

Serum enzymes in head and neck cancer. IV: 5-nucleotidase.

Serum 5-nucleotidase levels have been estimated in a group of 50 patients with head and neck cancer. The mean value was significantly higher in patients compared to the controls. In patients with non-malignant growths, the activity was comparable with the controls. The increase was higher in patients with proliferative lesions than those with ulcerative growths. Enzyme activity was found to be increased with the advancement in the stage of cancer. The rise was comparatively higher in patients with cervical metastasis. After radiotherapy, a gradual and significant reduction of serum 5-nucleotidase activity was observed.

5'-Nucleotidase↗

PubMed1984

5'-Nucleotidase activity and arachidonate metabolism in doxorubicin sensitive and resistant P388 cells.

5'-nucleotidase activity, arachidonate metabolism and adenosine uptake were measured in P388 murine leukaemia cells and in a subline resistant to doxorubicin. These membranes related activities were found to be increased in the doxorubicin resistant cell line, compared to the drug sensitive cells. It is suggested that these differences do not play a role in the mechanism of resistance to doxorubicin. Rather they reflect alterations in plasma membrane composition and structure between these cell lines. This study also suggests that the use of decreased 5'-nucleotidase activity as a marker of certain leukaemias should be reviewed with caution. An increase in cell enzyme activity in treated patients may not necessarily indicate a shift toward normal behaviour of these cells, but rather a selection of certain cell subpopulations.

5'-Nucleotidase↗

PubMed1982

Solubilization and purification of rat liver 5'-nucleotidase by use of a zwitterionic detergent and a monoclonal-antibody immunoadsorbent.

1. A variety of detergents were used to solubilize 5'-nucleotidase from rat liver plasma membranes. 2. The zwitterionic detergent Sulphobetaine 14 gave optimal solubilization by the criteria of release into a high-speed-centrifugation supernatant and the formation of the smallest and least polydisperse active enzyme observed on polyacrylamide-gel electrophoresis. 3. The Sulphobetaine 14-solubilized enzyme from rat liver was purified by using the conventional techniques of ion-exchange chromatography and gel filtration, or by an immunoaffinity step with a monoclonal antibody immunoadsorbent. 4. 5'-Nucleotidase was purified at least 12 000-fold relative to liver homogenate by the immunoaffinity purification scheme and had a specific activity in the range 285-340 mumol/min per mg of protein. The yield was in the range 9-16%. 5. The purified enzyme shows a major polypeptide band of apparent Mr 70 000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and a minor band of apparent Mr 38 000. 6. A rational approach to the general problem of the purification of minor intrinsic membrane proteins is discussed, with the use of polyacrylamide-gel electrophoresis to determine the most appropriate detergent and monoclonal antibodies in subsequent immunoaffinity purification.

5'-Nucleotidase↗

PubMed1984

The existence and properties of two dimers of rat liver ecto-5'-nucleotidase.

Immunoaffinity-purified rat liver 5'-nucleotidase contained two subunits of Mr 70 000 (alpha) and 38 000 (beta). Charge-shift electrophoresis and chemical cross-linking revealed that approx. 80% of the solubilized enzyme activity occurred as an alpha alpha-dimer of Mr 140 000. The remaining 20% was an alpha beta-dimer of Mr 108 000. The beta-subunit did not possess enzymic activity. Peptide mapping and immunoblotting with antibodies against the alpha- and beta-subunits showed that the beta-subunit was homologous with a part of the alpha-subunit. Three monoclonal antibodies against rat liver 5'-nucleotidase were characterized as binding to the extracellular domain of the enzyme. All three monoclonal antibodies and concanavalin A bound to the alpha-subunit, but no binding could be detected to the beta-subunit. It was therefore concluded that the beta-subunit was a fragment of an alpha-subunit that had lost an extracellular domain. Both forms of the enzyme occurred in freshly solubilized membrane preparations as well.

5'-Nucleotidase↗

PubMed1985

5'-Nucleotidase from rat heart membranes. Inhibition by adenine nucleotides and related compounds.

ADP and ATP and their analogues were evaluated as inhibitors of 5'-nucleotidase purified from heart plasma membrane. ADP analogues are more powerful inhibitors than the corresponding ATP analogues. The most powerful inhibitor found is adenosine 5'-[alpha beta-methylene]diphosphate (AOPCP) for which the enzyme shows a Ki of 5 nM at pH 7.2. Measurements of pKi values for ADP and AOPCP as a function of pH indicate that the major inhibitory species of both nucleotides is the dianion. In the physiological range of pH values, AOPCP is a more powerful inhibitor than ADP principally because a higher percentage of AOPCP exists in the dianion form. The methylenephosphonate analogue of AMP (ACP), though not a substrate, is a moderately effective inhibitor. The corresponding analogues of ADP (ACPOP) and ATP (ACPOPOP) are as good inhibitors as ADP and ATP respectively. The thiophosphate analogues of ADP all inhibit 5'-nucleotidase, although not as powerfully as ADP, the most effective of these analogues being adenosine 5'-O-(1-thiodiphosphate) diastereoisomer B (ADP[alpha S](B)]. Other nucleotides inhibit the enzyme, but none is as effective as AOPCP. Inorganic tripolyphosphate and methylenediphosphonate are better inhibitors of the enzyme than is inorganic pyrophosphate. Inorganic thiophosphate is a better inhibitor than is orthophosphate. Hill plots of the ADP and AOPCP inhibition yield slopes close to 1; Hill plots of the ATP inhibition yield slopes of about 0.6. MgADP- is not an inhibitor, and MgATP2- is at best a very weak inhibitor of the enzyme.

5'-Nucleotidase↗

PubMed1986

Regulation of rat heart cytosol 5'-nucleotidase by adenylate energy charge.

A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5'-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.

5'-Nucleotidase↗

PubMed1989

Purification and properties of a novel nucleotide-hydrolysing enzyme (5'-nucleotidase) from Boophilus microplus.

The tick Boophilus microplus contains a nucleoside phosphate-hydrolysing enzyme which, in many respects, resembles the well characterized 5'-nucleotidase from mammalian tissue. The tick enzyme has been purified to homogeneity. It is a membrane-bound glycoprotein with an apparent Mr of 67,000 and, although it fails to hydrolyse a range of nucleoside 2'- or 3'-monophosphates, it has broad specificity for the 5' derivatives. Further investigation of the enzyme's substrate specificity, however, shows some important differences from the mammalian nucleotidases. It hydrolyses both bis-p-nitrophenyl phosphate and p-nitrophenyl phenylphosphonate, typical substrates for phosphodiesterases. However, the tick enzyme is most strikingly different from the mammalian enzymes in that it hydrolyses not only AMP but ADP and ATP as well. Further, the products of the hydrolysis of ATP are adenosine and tripolyphosphate, a reaction which has not been reported previously. The products of ADP hydrolysis are adenosine and pyrophosphate.

5'-Nucleotidase↗

PubMed1984

Ferritin immunoelectron microscopic localization of 5'-nucleotidase on rat liver cell surface.

Rat livers were prefixed by perfusion with 0.6% glutaraldehyde and briefly homogenized with a Teflon-glass homogenizer. The prefixed cells isolated by low-speed centrifugation in high yield effectively preserved the original polygonal shape and polarity. These cells were incubated with ferritin-antibody conjugates monospecific for rat liver 5'-nucleotidase, and the localization of the enzymes on the surface of hepatocytes and endothelial cells was quantitatively investigated. It was revealed that the surface density of 5'-nucleotidase is much higher on the bile canalicular surface than on the sinusoidal surface and only a few ferritin particles were detected on the lateral surface. On the bile canalicular surface ferritin particles were almost exclusively found on the microvilli in larger clusters. Similar distribution was also observed on the sinusoidal surface but the size of cluster was much smaller. On both surfaces many fewer ferritin particles were found on the intermicrovillar region, including the coated pits region, than on the microvillar region. Ferritin particles were also found on the endothelial cell surface.

5'-Nucleotidase↗