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PubMed1989

5'-nucleotidase activity in prostatic carcinoma and benign prostatic hyperplasia.

In light of previous reports of alterations in 5'-nucleotidase activity in neoplastic conditions, 5'-nucleotidase activity was examined histochemically in tissue sections and quantified biochemically in extracts of human hyperplastic prostates and prostatic carcinomas obtained surgically. The 5'-nucleotidase activities per mg protein in extracts of 29 prostatic carcinomas were lower (P less than 0.0005) than in extracts from 10 samples of benign prostatic hyperplasia. The 5'-nucleotidase activity per mg protein in extracts of prostatic carcinoma from the 29 patients correlated (R = -0.369, P = 0.049) with the degree of histological differentiation; the extracts of poorly differentiated carcinomas contained low levels of 5'-nucleotidase. When age and histological differentiation (Gleason's grade) were adjusted, the enzyme activity per mg protein correlated (R = 0.242, P = 0.004) with patient survival. When all three parameters were considered together, i.e., histological grade of the tumor, 5'-nucleotidase extracted from the tumor, and age of the patient, they were found to be mutually complementary for the prediction of patient survival (R = 0.388, P = 0.0001). To our knowledge, this is the first report that prostatic epithelium expresses 5'-nucleotidase; further work will be required to define the reasons for the high levels of activity observed in prostates without cancer and for the decrease in the activity in prostatic carcinoma.

5'-Nucleotidase↗
PubMed1986

Biological variance of cholinesterase and 5'-nucleotidase in serum of healthy persons.

We measured cholinesterase (EC 3.1.1.8) and 5'-nucleotidase (EC 3.1.3.5) activities in serum of 24 healthy laboratory staff during 12 months. Overall mean activities ranged from 5.3 to 13.4 kU/L for cholinesterase and 5.4 to 9.8 U/L for 5'-nucleotidase. Cholinesterase activity was significantly (p less than 0.01) higher for men than for women. 5'-Nucleotidase activity was significantly (p = 0.01) higher for subjects 40 years or older than for those younger than 40, but was not different with respect to sex or time of year. Average intra- and interindividual variances (SD2) were 0.38 and 2.69 for cholinesterase and 1.41 and 0.97 for 5'-nucleotidase, respectively. Intra- to interindividual standard deviation ratios were 0.38 for cholinesterase and 1.21 for 5'-nucleotidase. Average within-run analytical variances were 0.13 and 0.3 (4% and 13% of total variance) for cholinesterase and 5'-nucleotidase, respectively. The importance of these findings in regards to diagnostic interpretation of serum cholinesterase and 5'-nucleotidase results is discussed.

5'-Nucleotidase↗
PubMed1998

Association of ecto-5'-nucleotidase with specific cell types in the adult and developing rat olfactory organ.

A unique feature of the olfactory epithelium is its ability to give rise to new sensory neurons throughout life and also following injury. Cells at the basal side of the epithelium serve as neurogenic progenitor cells. The enzyme ecto-5'-nucleotidase is expressed at the surface of developing nerve cells and is regarded as a marker of neural development. To study the expression pattern of the enzyme, we analyzed its distribution in the adult and developing rat olfactory organ. Labeling is restricted to specific cell types and varies between the epithelia investigated. At the basal side of the olfactory epithelium, activity of 5'-nucleotidase is associated specifically with the dark/horizontal basal cells. Neither the light/globose basal cells, which are the immediate precursors of the sensory receptor cells, nor subsets of potentially immature olfactory receptor cells are labeled. On the other hand, microvillar cells dispersed at the lumenal side of the epithelium contain 5'-nucleotidase activity. The enzyme is also present at the inner lining of the ducts of Bowman's glands as they traverse the epithelium. Within the respiratory epithelium, activity of 5'-nucleotidase is associated with basal cells as well as with the epithelial surface. During development, 5'-nucleotidase is initially limited to the respiratory epithelium, including its basal cells. Dark/horizontal basal cells of the olfactory epithelium, which are positive for 5'-nucleotidase, first appear at the border of the respiratory epithelium, suggesting that they might originate from immigrating basal cells of the respiratory epithelium. Within the vomeronasal organ, labeling is largely restricted to the receptor-free epithelium. Although the functional role of 5'-nucleotidase in the olfactory system needs to be further defined, the distribution of the enzyme can be used successfully as a marker for defined cell types.

5'-Nucleotidase↗
PubMed1994

Distribution of ecto-5'-nucleotidase in the rat liver: effect of anaemia.

In the kidney a striking parallel exists between the expression of ecto-5'-nucleotidase and of erythropoietin by renal fibroblasts. It was therefore hypothesized that the expression of ecto-5'-nucleotidase in fibroblasts might be controlled by oxygen tension. In order to test this hypothesis, we examined the distribution of the enzyme in a tissue which displays a defined zonation in respect to oxygen tension, namely in the liver; anaemia was used in order to exaggerate this zonation. The distribution of ecto-5'-nucleotidase was investigated by light and electron microscopy using enzyme and immunohistochemical methods in the livers of healthy and of anaemic rats. Anaemia was produced by haemolysis combined with X-ray irradiation. The enzyme was detected in the bile canaliculi, in the connective tissue of the portal triads and of the central veins, and in fat-storing cells probably corresponding to a special form of fibroblasts. In healthy animals the perisinusoidal ecto-5'-nucleotidase activity was slightly higher in the pericentral than in the periportal area of the acinus whereas the inverse was observed for the staining of bile canaliculi. Anaemia provoked an increase of ecto-5'-nucleotidase in fat-storing cells in the pericentral zone of the acinus and in fibroblasts around the central veins, resulting in steepended gradients along the sinusoids. The intralobular gradient of ecto-5'-nucleotidase in perisinusoidal cells and the effect thereon of anaemia suggest that the expression of the ecto-5'-nucleotidase might be directly or indirectly controlled by local oxygen tension.

5'-Nucleotidase↗
PubMed1995

5'-nucleotidase activity as a synaptic marker of parasagittal compartmentation in the mouse cerebellum.

In the molecular layer of the mouse cerebellum, the histochemical activity of the adenosine-producing ectoenzyme 5'-nucleotidase discloses a parasagittal pattern of alternating enzyme-rich and enzyme-poor bands. In the rat, 5'-nucleotidase activity transiently labels cerebellar synapses during postnatal development and shifts later on towards an exclusive glial location in the molecular layer. We therefore asked whether different ultrastructural expression of 5'-nucleotidase would account for the light microscopic pattern seen in the adult mouse cerebellum. Using an enzyme cytochemical method, we localized 5'-nucleotidase activity on the glial cells and at the main types of asymmetrical synapses in the developing and mature cerebellum of the mouse. The percentage of labelled synapses increased until adulthood within the 5'-nucleotidase-positive bands. Here, the vast majority (86%) of the synapses were labelled against only 27% within the negative bands in the adult. Thus, 5'-nucleotidase appears as a marker of glia and of Purkinje cell synapses across cerebellar compartments. Changes in purinergic neuromodulation and/or cell adhesion mediated by 5'-nucleotidase across bands might participate in the functional differentiation of the cerebellar parasagittal subsets.

5'-Nucleotidase↗
PubMed1995

Ecto-5'-nucleotidase is expressed by pericytes and fibroblasts in the rat heart.

Ecto-5'-nucleotidase is anchored at the outer surface of cell membranes and thus its reaction product adenosine is released into the extracellular space. Extracellular adenosine displays via specific receptors a wide range of physiological effects in heart. There are discrepancies in the literature concerning the distribution of ecto-5'-nucleotidase in heart. Since we suspected that these may be due to technical problems, in the present study on ecto-5'-nucleotidase in rat heart we attempted to circumvent some technical pitfalls. Good preservation of the tissue with open capillary lumina, providing a clear identification of endothelium, was obtained by perfusion fixation. At the light microscopic level, the distribution of ecto-5'-nucleotidase studied by enzyme histochemistry and immunohistochemistry using a monoclonal and a polyclonal antibody yielded congruent results. The enzyme was rather homogeneously distributed throughout the myocardium, with a slightly higher incidence of stained cells in the outer thirds than in the inner third of the wall. Consistently high levels of ecto-5'-nucleotidase were seen only in interstitial cells. The walls of large vessels and heart muscle cells were constantly negative for ecto-5'-nucleotidase. The endothelia of capillaries were mostly negative but a few profiles occasionally displayed a weak immunoreaction. The interstitial cells staining positive for ecto-5'-nucleotidase could be identified as pericytes and as fibroblasts according to their shapes and localizations. The immunoreactivity of fibroblasts was confirmed by electron microscopy. These data indicate that adenosine may be formed extracellularly in the interstitium of the myocardium, where it would have direct access to important targets such as myocytes, arterioles and nerve endings.

5'-Nucleotidase↗
PubMed1975

Purification and characterization of a cyclic nucleotide-regulated 5'-nucleotidase from potatoe.

A procedure is presented for the rapid purification of a 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) from potato tubers, involving ammonium sulphate fractionation and chromatography on phosphocellulose, DEAE-cellulose and Sephadex G-75. Application of this procedure results in a 6000-fold purification of the 5'-nucleotidase and the final preparations are virtually homogeneous, yielding only one protein band on electrophorsis in polyacrylamide gels in non-dissociating or dissociating conditions. The 5'-nucleotidase has a molecular weight of 50 000 from gel filtration experiments. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the purified 5'-nucleotidase reveals one major band of molecular weight 25 000. The 5'-nucleotidase is competitively inhibited by cyclic nucleotides, having micromolar Ki values for cyclic AMP and cyclic GMP at pH 5.0 and pH 8.0. The enzyme has a pH optimum of 5.0 with 5'-GMP as substrate. While 5'-AMP and 3'-AMP are hydrolyzed at comparable rates at pH 5.0, at pH 8.0 the rate of hydrolysis of 3'-AMP is only 4% of that with 5'-AMP. ADP, ATP and 2'-AMP are very poor substrates for the enzyme. The nucleotidase has micromolar Km values for nucleoside 5'-monophosphates other than 5'-NMP. A wide variety of divalent cations activate the 5'-nucleotidase.

Cadmium↗
PubMed1977

Control of the production and partial characterization of repressible extracellular 5'-nucleotidase and alkaline phosphatase in Neurospora crass.

A new species of orthophosphate repressible extracellular 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was found to be released into mycelial culture media when a wild type strain of Neurospora crassa was grown on limiting amounts of phosphate. The production of 5'-nucleotidase and extracellular acid and alkaline phosphatase was inhibited by the addition of rifampicin when it was added at the later stage of mycelial growth, but not when it was added at a very early stage. The 5'-nucleotidase and extracellular alkaline phosphatase were partially purified and characterized. pH optimum of the former was 6.8 and that of the latter was higher than 10.0. The 5'-nucleotidase activity was inhibited by ethylenediaminetetraacetate (EDTA) and ZnCl2 at pH 6.8 and stimulated by MnCl2 and CoCl2 at pH 4.0. Alkaline phosphatase activity was stimulated by EDTA, MgCl2, CoCl2 and MnCl2. 5'-nucleotidase activity was stimulated by EDTA, MgCl2, CoCl2 and MnCl2. 5'-nucleotidase hydrolyzed various 5'-nucletides but not 3'-nucleotides or other various phosphomono- and diester compounds. Alkaline phosphatase hydrolyzed all the phosphomonoester compounds tested. Mutants, nuc-1 and nuc-2, which were originally isolated by the inability to utilize RNA or DNA as a sole source of phosphate, were unable to produce 5'-nucleotidase or six other repressible enzymes reported previously. These mutants showed no or significantly reduced growth on orthophosphate-free nucleotide media depending on the number of conidia inoculated, mainly because of loss of ability to produce these repressible extracellular phosphatases.

Alkaline Phosphatase↗
PubMed1991

Isolation and characterization of 5'-nucleotidase of a human pancreatic tumor cell line.

5'-Nucleotidase of a human pancreatic tumor cell line (PaTu II) has been purified to homogeneity after extraction with detergent followed by two affinity chromatographic steps. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified 5'-nucleotidase revealed a single polypeptide band of 67 kDa. The Western blotted enzyme can be overlaid with concanavalin A proving its glycoprotein nature. After treatment with endoglycosidase F the deglycosylated 5'-nucleotidase exhibits an apparent molecular mass of 58 kDa. The kinetic properties of the solubilized enzyme have been determined (Km (AMP) of 4.0 microM; Vmax (AMP) = 8.6 muMOL/min.mg). Adenosine 5'-[alpha,beta-methylene]diphosphate is a competitive inhibitor of 5'-nucleotidase, whereas concanavalin A inhibits the enzymatic activity in a non-competitive manner. Polyclonal antibodies against purified 5'-nucleotidase of PaTu II have been produced which inhibit its enzymatic activity. Polyclonal antibodies raised against the enzyme purified from rat liver or bull seminal plasma also recognize 5'-nucleotidase of PaTu II cells, whereas polyclonal and monoclonal antibodies against the enzyme derived from chicken gizzard show no cross-reactivity. 5'-Nucleotidase appears to be concentrated in the plasma membrane of PaTu II cells as judged by cell fractionation and indirect immunofluorescence studies.

5'-Nucleotidase↗
PubMed1997

Focal cerebral ischemia enhances glial expression of ecto-5'-nucleotidase.

The effect of ischemia on the reactive expression of ecto-5'-nucleotidase in rat brain was studied 6 h and 1, 2 and 7 days after permanent middle cerebral artery occlusion (MCAO). The distribution of 5'-nucleotidase in the infarcted brain was compared to markers for astrocytes (glial fibrillary acidic protein (GFAP)) and microglia (complement receptor type 3, antibody OX42) using histological staining or immunohistochemistry. 5'-Nucleotidase could be associated with reactive astrocytes by immunohistochemistry and with reactive microglia by enzyme histochemistry. In the untreated control 5'-nucleotidase was associated with astrocytes only in the hippocampus and the submeningeal space. After ischemia the enzyme was expressed on reactive astrocytes in the tissue surrounding the volume of infarction. Individual reactive astrocytes were observed 6 h after MCAO and the astrocytic expression became continuously enhanced during the following days. An enzyme histochemical analysis of 5'-nucleotidase activity revealed a postischemic increase in reaction product around the infarcted tissue. Seven days after MCAO a discrete band (0.2-0.4 mm) of reaction product characterized the rim of the infarcted area. This band of activity of 5'-nucleotidase colocalized with a band of immunoreactivity for OX42, indicative of an intense accumulation of 5'-nucleotidase expressing microglia. Our results suggest that ischemia following permanent MCAO results in an upregulation of the capacity for the hydrolysis of nucleotides within the tissue adjacent to the infarcted volume. Nucleotides released from the damaged cells can be hydrolyzed and the adenosine eventually formed may exert neuroprotective functions limiting the extent of damage.

5'-Nucleotidase↗
PubMed1999

Tyramine produces interstitial adenosine-mediated activation of ecto-5'-nucleotidase in rat heart in vivo.

We examined the effect of tyramine on the production of adenosine in rat heart. A flexibly mounted microdialysis setup was used to measure the concentration of interstitial adenosine and to assess the activity of ecto-5'-nucleotidase in in vivo rat hearts. The microdialysis probe was implanted in the left ventricular myocardium of anesthetized rats and perfused with Tyrode solution containing adenosine 5'-monophosphate (AMP) at a rate of 1.0 microl/min. The concentration of adenosine in the effluent (dialysate) was measured by high-performance liquid chromatography (HPLC). Dialysate adenosine obtained during perfusion with the AMP-containing solution through the probe originated from the hydrolysis of AMP by endogenous ecto-5'-nucleotidase, and the level of adenosine reflected the activity of ecto-5'-nucleotidase in the tissue. Tyramine (0-4 mM) increased the adenosine concentration measured during the perfusion of AMP (100 microM) in a concentration-dependent manner. Alpha,beta-methyleneadenosine 5'-diphosphate (alpha,beta-meADP, 100 microM), an inhibitor of ecto-5'-nucleotidase, abolished the AMP-induced increase in dialysate adenosine. Tyramine (1 mM) increased the adenosine concentration measured in the presence of 100 microM AMP (i.e., the activity of ecto-5'-nucleotidase) by 65.8 +/- 19.9% (n = 6, P < 0.05), an increase which was inhibited by an antagonist of the alpha1-adrenoceptor (prazosin, 50 microM) or of protein kinase C (chelerythrine, 10 microM). These data provide the first evidence that alpha1-adrenoceptor stimulation and the subsequent activation of protein kinase C can increase adenosine concentrations in the interstitial space of ventricular muscle in vivo, through activation of endogenous ecto-5'-nucleotidase. To examine the effect of tyramine on the production of adenosine by ischemia-reperfusion of the rat myocardium, the heart was subjected to myocardial ischemia for 15 min by occlusion of the left anterior descending coronary artery. When the heart was reperfused, elevation of the level of adenosine in the ischemic zone was observed, but this change was not significant. However, when corresponding experiments were performed with a subsequent systemic administration of tyramine (1 mM), a marked elevation in the level of adenosine was observed. The results suggest that tyramine elevates adenosine via stimulation of alpha1-adrenoceptors and protein kinase C-mediated activation of ecto-5'-nucleotidase in rat heart.

5'-Nucleotidase↗
PubMed1990

Cytochemistry of 5'-nucleotidase in the superior cervical ganglion of the rat: effects of pre- and postganglionic axotomy.

5'-Nucleotidase, a prominent nucleoside-producing ectoenzyme of glial plasma membranes, was studied by enzyme cytochemistry in the superior cervical ganglion of the rat under normal conditions and after pre- and postganglionic axotomy. In normal ganglia 5'-nucleotidase was restricted to capillary endothelial cells, localized both on the luminal surface and in pinocytotic vesicles. Following preganglionic axotomy, the number of enzyme-positive endothelial vesicles increased, whereas no 5'-nucleotidase was found on reactive Schwann cells during phagocytosis of degenerating preganglionic axon terminals. After postganglionic axotomy an even stronger increase in enzyme-containing endothelial vesicles occurred. In addition, 5'-nucleotidase activity became detectable on the plasma membrane of Schwann cells and proliferating satellite cells, which participate in the detachment of synapses from axotomized neurons. Fibroblasts in the endoneuronal connective tissue of regenerating ganglia also exhibited 5'-nucleotidase on their surface. The results obtained suggest that 5'-nucleotidase may be related to specific metabolic requirements of Schwann and satellite cells during regeneration and that these requirements differ from those of reactive Schwann cells after denervation of the ganglion. Postoperative changes in 5'-nucleotidase activity on endothelial cells and fibroblasts of the ganglion indicate an involvement of these cells in metabolic response elicited by pre- or postganglionic axotomy.

5'-Nucleotidase↗
PubMed1998

Behaviour of human lymphocytic isoenzymes of 5'-nucleotidase.

The behaviour of 5'-nucleotidase isoenzymes (ecto-5'-nucleotidase, e-Ns and c-N-II soluble 5'-nucleotidases) was studied in lymphocytes from patients with B-cell chronic lymphocytic leukemia. A strong reduction in ecto- and soluble activities was observed, although the pattern of the three 5'-nucleotidases did not always strictly overlap. A significant decrease (p<0.05) in ecto-5'-nucleotidase, e-Ns and c-N-II was found in B and T populations (B lymphocytes: 1.13, 0.88 and 1.26 nmol/h/10(6) cells versus 95.96, 9.64 and 13.73 nmol/h/10(6) cells in controls; T lymphocytes: 1.31, 0.23 and 0.06 nmol/h/10(6) cells versus 9.25, 1.31 and 2.10 nmol/h/10(6) cells in healthy subjects). The percentage of ecto-5'-nucleotidase-positive cells (CD73+) was reduced in leukemia patients, indicating a lower number of active molecules on the cell surface. The results of RT-PCR analysis showed that the ecto-5'-nucleotidase mRNA of leukemia patients was not defective.

5'-Nucleotidase↗
PubMed2003

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat.

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.

5'-Nucleotidase↗
PubMed2006

Lymphocyte ecto-5'-nucleotidase in obese type 2 diabetic patients treated with gliclazide.

OBJECTIVE: Lymphocyte 5'-nucleotidase is sensitive to superoxide anion, and is an indicator of oxidative stress in humans. The aim of this study was to assess the effect of the sulfonylurea drugs gliclazide and glibenclamide on lymphocyte ecto-5'-nucleotidase of type 2 diabetic patients. METHODS: Thirty obese type 2 diabetic patients were treated for three months after randomisation either with gliclazide or glibenclamide. Basic laboratory parameters (glycaemia, fructosamine, C-peptide), plasma malondialdehyde levels (MDA) as well as lymphocyte 5'-nucleotidase activity were determined, for all patients and 16 healthy controls, before and after the treatment. RESULTS: 5'-nucleotidase activity in diabetic patients before treatment with gliclazide was 1.61 +/- 0.16 nmol/min/10(6) lymphocytes, and was significantly (P < 0.01) increased compared with the level in healthy controls. After three months of gliclazide treatment, ecto-5'-nucleotidase activity fell significantly by 47.39% and 36% in unstimulated Con A- and PMA-stimulated lymphocytes, respectively. Glibenclamide treatment had no effect on ecto-5'-nucleo-tidase of type 2 diabetic patients. Glycoregulation was improved, as plasma fructosamine decreased from 53.4 to 42.1 and from 50.5 to 43.4 U/g proteins after gliclazide and glibenclamide treatment, respectively. Plasma MDA levels markedly decreased after gliclazide but not glibenclamide treatment. CONCLUSION: These results show that gliclazide treatment inhibits the activity of lymphocyte ecto-5'-nucleotidase and presumably de-creases the concentration of adenosine at the cell surface. A decrease in 5'-nucleotidase activity and attenuation of adenosine production may be a factor in the protection of tissue injury in type 2 diabetic patients.

5'-Nucleotidase↗
PubMed1990

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.

5'-Nucleotidase↗
PubMed1998

Release of the glycosylphosphatidylinositol-anchored enzyme ecto-5'-nucleotidase by phospholipase C: catalytic activation and modulation by the lipid bilayer.

Many hydrolytic enzymes are attached to the extracellular face of the plasma membrane of eukaryotic cells by a glycosylphosphatidylinositol (GPI) anchor. Little is currently known about the consequences for enzyme function of anchor cleavage by phosphatidylinositol-specific phospholipase C. We have examined this question for the GPI-anchored protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5), both in the native lymphocyte plasma membrane, and following purification and reconstitution into defined lipid bilayer vesicles, using Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC). Membrane-bound, detergent-solubilized and cleaved 5'-nucleotidase all obeyed Michaelis-Menten kinetics, with a Km for 5'-AMP in the range 11-16 microM. The GPI anchor was removed from essentially all 5'-nucleotidase molecules, indicating that there is no phospholipase-resistant pool of enzyme. However, the phospholipase was much less efficient at cleaving the GPI anchor when 5'-nucleotidase was present in detergent solution, dimyristoyl phosphatidylcholine, egg phosphatidylethanolamine and sphingomyelin, compared with the native plasma membrane, egg phosphatidylcholine and a sphingolipid/cholesterol-rich mixture. Lipid molecular properties and bilayer packing may affect the ability of PI-PLC to gain access to the GPI anchor. Catalytic activation, characterized by an increase in Vmax, was observed following PI-PLC cleavage of reconstituted 5'-nucleotidase from vesicles of several different lipids. The highest degree of activation was noted for 5'-nucleotidase in egg phosphatidylethanolamine. An increase in Vmax was also noted for a sphingolipid/cholesterol-rich mixture, the native plasma membrane and egg phosphatidylcholine, whereas vesicles of sphingomyelin and dimyristoyl phosphatidylcholine showed little activation. Km generally remained unchanged following cleavage, except in the case of the sphingolipid/cholesterol-rich mixture. Insertion of the GPI anchor into a lipid bilayer appears to reduce the catalytic efficiency of 5'-nucleotidase, possibly via a conformational change in the enzyme, and activity is restored on release from the membrane.

5'-Nucleotidase↗
PubMed1999

Human high-Km 5'-nucleotidase effects of overexpression of the cloned cDNA in cultured human cells.

5'-Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism. Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high-Km nucleotidases and 5'(3')-nucleotidases. Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y. & Inoue, S. (1994) Biochem. Biophys. Res. Commun. 205, 917-922) we cloned the complete cDNA of the human high-Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells. After transfection, E. coli overproduced large amounts of the enzyme. Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein. Part of the enzyme, corresponding to approximately 2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtained with a vector where the 3'-end of the nucleotidase coding sequence is linked to the 5'-end of the green fluorescent protein coding sequence. Several green clones overproduced both mRNA and fusion protein. Two clones with 10-fold higher enzyme activity were analyzed further. The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high-Km enzyme. The growth rate of the two clones did not differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation.

5'-Nucleotidase↗