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PubMed1995

Evidence that 5'-nucleotidase is associated with malleable synapses--an enzyme cytochemical investigation of the olfactory bulb of adult rats.

The adenosine-producing ecto-enzyme 5'-nucleotidase has recently been assigned to malleable axon terminals in both the developing and regenerating adult nervous system, but is otherwise only glia-bound. Using a cytochemical lead method, we now show that 5'-nucleotidase activity is localized predominantly at glomerular and mitral synapses within the main olfactory bulb of normal, adult rats. As these terminals are prone to synaptic turnover even at maturity, the present findings favour the view that this enzyme constitutes a marker molecule for plastic synapses. It is suggested that functions of 5'-nucleotidase in purinergic neuromodulation and cell adhesion are unique to the olfactory bulb, and implied in synaptic arrangements and information processing.

Animals↗

PubMed1995

A1-receptor-mediated effect of adenosine on the release of acetylcholine from the myenteric plexus: role and localization of ecto-ATPase and 5'-nucleotidase.

No attempt has been made so far to classify the subtypes of presynaptic inhibitory adenosine receptors located in the myenteric plexus and to localize ecto-ATPase and 5'-nucleotidase in the intestine. The release of [3H]acetylcholine and smooth muscle responses to acetylcholine were measured and the effect of selective adenosine receptor ligands was studied using field-stimulated isolated longitudinal muscle strips of guinea-pig ileum. Release of ATP and its hydrolysis rate were also measured using the luciferin-luciferase technique. A histochemical method combined with electron microscopy was used for localization of ecto-ATPase and 5'-nucleotidase, enzymes responsible for destruction of extracellular ATP, ADP and AMP. Subtype-selective A1-receptor agonists and antagonists inhibited and enhanced, respectively, the release of acetylcholine associated with neuronal activity. A significant amount of ATP was released in response to electrical stimulation and administration of carbamylcholine. The release of ATP was inhibited by atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3-receptor antagonist. Hydrolysis of ATP was rapid and resulted in an accumulation of extracellular adenosine involved in presynaptic A1-receptor-mediated inhibition of acetylcholine release. While the inhibitory effect of adenosine and ATP was significantly potentiated by dipyridamol, an adenosine uptake blocker, that of 2-ms ATP was not. The effect of ATP was not competitively antagonized by 8-cyclopentyl-1,3-dipropylxanthine, a selective A1-receptor antagonist. In conclusion, axon terminals of cholinergic interneurons are equipped with inhibitory A1- and P2 gamma-receptors. Therefore, both adenosine and ATP control the release of acetylcholine through these receptors. ATP is mainly released from the smooth muscle in response to stimulation of M3-muscarinic receptors by endogenous acetylcholine (cascade transmission [Vizi E. S. et al. (1992) Neuroscience 50, 455-465]) and is rapidly hydrolysed by ecto-ATPase localized on the surface of the smooth muscle and axon terminals producing ADP and AMP, and by 5'-nucleotidase present only on the surface of smooth muscle cells producing adenosine.

5'-Nucleotidase↗

PubMed1993

The nucleotidase of Boophilus microplus and its relationship to enzymes from the rat and Escherichia coli.

Boophilus microplus contains a nucleotidase-like enzyme which is able to hydrolyze a range of nucleoside 5'-mono-, di- and triphosphates to the nucleoside. Its relationship to several other nucleotide hydrolyzing enzymes has been explored. Limited peptide sequencing shows similarities to both mammalian nucleotidases and the Escherichia coli uridine diphosphate sugar hydrolase. The tick enzyme also hydrolyzes UDP-glucose, though by a mechanism different to that of the bacterial enzyme. On the other hand, it resembles the mammalian nucleotidases in that there is evidence that it is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor.

5'-Nucleotidase↗

PubMed2006

Ecto-nucleotidases of the CD39/NTPDase family modulate platelet activation and thrombus formation: Potential as therapeutic targets.

Extracellular nucleotide P2-receptor-mediated effects on platelets, leukocytes and endothelium are modulated by ecto-nucleotidases. These ecto-enzymes hydrolyze extracellular nucleotides to the respective nucleosides. The dominant ecto-nucleotidase expressed by the endothelium, by monocytes and vascular smooth muscle cells is CD39/NTPDase1. Ecto-nucleotidase biochemical activity of CD39 is lost at sites of acute vascular injury, such as in ischemia reperfusion and immune graft rejection. CD39L(Like)1/NTPDase2, a related protein, is associated with the basolateral surface of endothelium, the adventitia of vessels and microvascular pericytes. CD39/NTPDase1 hydrolyzes both tri- and diphosphonucleosides and blocks platelet aggregation responses to ADP. In contrast, CD39L1/NTPDase2, a preferential nucleoside triphosphatase, activates platelets by preferentially converting ATP to ADP, the major agonist of platelet P2 receptors. Spatial and temporal expression of NTPDases in the vasculature appears to control platelet activation, thrombus size and stability by regulating phosphohydrolytic activity and consequent P2 receptor signaling. Constitutively circulating microparticles appear to be associated with functional NTPDases, and accumulation of these at sites of vascular injury might influence local thrombus formation and evolution. The phenotype of the cd39-null mouse is in keeping with disordered thromboregulation with heightened susceptibility to inflammatory vasculary reactions, increased permeability and high levels of tissue fibrin. Paradoxically, these mutant mice also exhibit a bleeding phenotype with differential platelet P2Y1 desensitization. Over-expression of CD39 at sites of vascular injury and inflammation by adenoviral vectors, by transgenesis or by the use of pharmacological modalities with soluble derivatives has been shown to have major potential in several animal models tested to date. Future clinical applications will involve the development of new therapeutic strategies to various inflammatory vascular diseases and in transplantation.

Animals↗

PubMed2004

Ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio).

Adenosine, a well-known neuromodulator, may be formed intracellularly in the CNS from degradation of AMP and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. This study reports the enzymatic properties of an ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for AMP hydrolysis in a pH range of 7.0-7.5 in the presence of Mg(2+). The enzyme presented a maximal activity for AMP hydrolysis at 37 degrees C. The apparent K(m) and V(max) values for Mg(2+)-AMP were 135.3+/-16 microM and 29+/-4.2 nmol Pi.min(-1).mg(-1) protein, respectively. The enzyme was able to hydrolyze both purine and pyrimidine monophosphate nucleotides, such as UMP, GMP and CMP. Levamisole and tetramisole (1 mM), specific inhibitors of alkaline phosphatases, did not alter the enzymatic activity. However, a significant inhibition of AMP hydrolysis (42%) was observed in the presence of 100 microM alpha,beta-methylene-ADP, a known inhibitor of ecto-5'-nucleotidase. Since 5'-nucleotidase represents the major enzyme responsible for the formation of extracellular adenosine, the enzymatic characterization is important to understand its role in purinergic systems and the involvement of adenosine in the regulation of neurotransmitter release.

5'-Nucleotidase↗

PubMed2005

Role of 5'-nucleotidase in thiopurine metabolism: enzyme kinetic profile and association with thio-GMP levels in patients with acute lymphoblastic leukemia during 6-mercaptopurine treatment.

Thiopurines are used for treatment of several diseases. Cytotoxicity is caused by the derived compounds 6-thioguanine nucleotides (TGNs) and methyl-6-thioinosine monophosphate (methylthio-IMP). The 6-thiopurine mononucleotides 6-thio-IMP (thio-IMP), 6-thio-GMP (thio-GMP) and methylthio-IMP can be catabolized by purine 5'-nucleotidase. It has been shown that the various 5'-nucleotidases are key enzymes for (6-thio)-purine metabolism. We aimed to investigate whether the overall 5'-nucleotidase (5'NT) activity is correlated with the efficacy and toxicity of 6-thiopurine nucleotides. Substrate affinity of 5'NT for IMP, GMP, AMP, thio-IMP, thio-GMP and methylthio-IMP was studied in human lymphocytes. For each of the substrates, the pH for optimal overall enzyme activity has been determined at a pH range between 6 and 10. At the optimal pH, assays were performed to establish Km and Vmax values. Optimal pH values for the various substrates were between 7 and 8.5. Km values ranged from 33 to 109 microM, Vmax ranged from 3.99 to 19.5 nmol/10(6) peripheral mononuclear cells (pMNC) h, and Vmax/Km ratios ranged from 105 to 250. The results did not show a distinct preference of 5'NT activity for any of the tested thiopurine nucleotides. The enzyme kinetic studies furthermore revealed substrate inhibition by thio-IMP and thio-GMP as a substrate. Inhibition by thio-GMP also seems to occur in patients treated with 6-mercaptopurine (6 MP); subsequently, this may lead to toxicity in these patients.

5'-Nucleotidase↗

PubMed2006

Methanol alters ecto-nucleotidases and acetylcholinesterase in zebrafish brain.

Methanol is a neurotoxic compound that is responsible for serious damage on CNS. Besides being found as an environmental contaminant, this alcohol is also employed as a component of cryoprotector solutions for zebrafish embryos. Here we tested the acute effect of methanol on ecto-nucleotidase (NTPDase, ecto-5'-nucleotidase) and acetylcholinesterase (AChE) activities in zebrafish brain. After acute treatment, there were significant decreases on ATP (26% and 45%) and ADP hydrolysis (26% and 30%) at 0.5% and 1.0%, respectively. However, no significant alteration on ecto-5'-nucleotidase activity was verified in zebrafish brain. A significant inhibition on AChE activity (39%, 33% and 30%) was observed at the range of 0.25% to 1.0% methanol exposure. Four NTPDase sequences were identified from phylogenetic analyses, which one is similar to NTPDase1 and the others to NTPDase2. Methanol was able to inhibit NTPDase1, two isoforms of NTPDase2 and AChE transcripts. To evaluate if methanol affects directly these enzymes activities, we have performed in vitro assays. ATP hydrolysis presented a significant inhibition (19% and 34%) at 1.5% and 3.0%, respectively, and ADP hydrolysis decreased only at 3.0% (29.2%). Nevertheless, AMP hydrolysis and AChE were not altered after in vitro exposure. The inhibitory effect observed on these enzymes could contribute to the neurodegenerative events promoted by methanol in zebrafish brain.

5'-Nucleotidase↗

PubMed1978

Regulation of purine biosynthesis in cultured Drosophila melanogaster cells: I.--Conditional activity of hypoxanthine-guanine-phosphoribosyltransferase and 5-nucleotidase.

In cultured cells established from Drosophila melanogaster embryos, and grown in usual medium, no hypoxanthine-guanine-phosphoribosyl transferase (HG-PRT) could be measured, and only traces of 5'-nucleotidase activity were detectable. On the contrary, it was observed that if the same medium is supplemented with purine bases, nucleosides, orotate, glutamine, azaserine or antifolates, de novo purine biosynthesis is inhibited, and HGPRT is detectable, along with an important 5'-nucleotidase activity. Moreover, dialysis or treatment of extracts from cells untreated by purines, with activated charcoal restored HGPRT and 5'-nucleotidase activities. These activities were abolished completely by inosinic acid (IMP) and guanosine 5'-monophosphoric acid (GMP). Similar results were obtained with fly extracts. These results suggest that de novo purine biosynthesis masks HGPRT activity, the endogenous synthesis leading to the accumulation of purine nucleotides which are inhibitors of the HGPRT activity.

Animals↗

PubMed1996

Effect of rapid eye movement sleep deprivation on 5'-nucleotidase activity in the rat brain.

Adenosine has been implicated in the regulation of rapid eye movement sleep (REMS). In an attempt to understand the mechanism of production of adenosine in relation to REMS it was hypothesized that should it be involved in REMS, the latter's deprivation is likely to affect its synthetic machinery. Hence, male albino rats were deprived of REMS by the flower pot technique and the activity of 5'-nucleotidase, an enzyme responsible for adenosine synthesis, was estimated in the cerebrum, cerebellum and brain stem. Suitable control experiments were conducted to rule out the non-specific effects. The results showed that 5'-nucleotidase activity decreased only after 4 days deprivation and in the cerebrum only; while short-term (2 days) deprivation did not affect the enzyme activity in any of the brain areas. The altered enzyme activity returned to baseline level after recovery from REMS deprivation. The results from other control experiments suggested that the effects were primarily due to REMS deprivation and not due to non-specific factors. It is proposed that if adenosine is involved in REMS, its production is unlikely to depend on 5'-nucleotidase or it may account primarily for EEG desynchronization.

5'-Nucleotidase↗

PubMed1985

Assay of human erythrocyte pyrimidine and deoxypyrimidine 5'-nucleotidase by isocratic reversed-phase high-performance liquid chromatography.

We report a rapid and reproducible assay for activity of human erythrocyte pyrimidine 5'-nucleotidase and deoxypyrimidine 5'-nucleotidase. The nucleotides CMP, UMP, dUMP, dCMP or dTMP are individually incubated 30 min at 37 degrees C with erythrocyte hemolysate and 4 mM magnesium chloride in Tris, pH 7.5. Data are provided for standardization of the reaction with each substrate. Individual nucleoside products are assayed in less than 10 min by reversed-phase high-performance liquid chromatography at 280 nm with 0-14% methanol in 0.01 M potassium dihydrogen phosphate. This is the first report of a high-performance liquid chromatographic assay system which allows quantitation of the activity of pyrimidine 5'-nucleotidase isozymes using five individual pyrimidine and deoxypyrimidine nucleotides as the substrates.

Chromatography, High Pressure Liquid↗

PubMed1998

The pharmacology and kinetics of ecto-nucleotidases in the perilymphatic compartment of the guinea-pig cochlea.

This study investigated the characteristics of ecto-nucleotidases in tissues lining the perilymphatic cavity of the cochlea. The perilymphatic space of the isolated guinea-pig cochlea was maintained with oxygenated artificial perilymph (AP) perfused at a rate of 100 microl/min. Following AP perfusion, either adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP) was introduced into scala tympani, and perfusion arrested for 2 min for substrate incubation with cochlear tissues. Effluent collected from the cochlea was assayed for adenine nucleotide metabolites by reverse-phase high-performance liquid chromatography (RP-HPLC). Extracellular ATP and ADP were rapidly and sequentially hydrolysed to adenosine by Ca2+/Mg2+-dependent and Ca2+/Mg2+-independent enzymatic mechanisms. The degradation of extracellular ATP, ADP and AMP occurred in the presence of intact tissues, as demonstrated by the limited lactate dehydrogenase (LDH) activity (0-2.2%). ATPase activity was not affected by inhibitors of intracellular ATPases (oligomycin, ouabain, N-ethylmaleimide, 100 microM NaN3) and non-specific alkaline phosphatase (beta-glycerophosphate). The hydrolysis of ATP was inhibited by 5 mM NaN3, suramin, ATPgammaS, La3+ and CTP, the hydrolysis of ADP by beta,gamma-imidoATP, and AMP degradation by alpha,beta-methyleneADP. Ecto-ATPase, ecto-ADPase and ecto-5'-nucleotidase followed Michaelis-Menten hyperbolic kinetics, with estimated Km values of 2282 microM, 6619 microM and 881 microM, respectively. Our results indicate the presence of considerable ecto-nucleotidase activity within scala tympani of the cochlea, and support its role as the terminating mechanism for P2 receptor signalling known to occur in the cochlea. A competition plot is consistent with ATP and ADP degradation mediated by the same enzyme (ecto-ADP diphosphohydrolase) with two different catalytic sites.

5'-Nucleotidase↗

PubMed2004

Effect of subchronic treatment with mercury chloride on NTPDase, 5'-nucleotidase and acetylcholinesterase from cerebral cortex of rats.

The aim of the present investigation was to evaluate the effect of a subchronic treatment (30 days/30 doses) with subcutaneous injections (0.1 mg/kg) of HgCl2 on NTPDase (E.C. 3.6.1.5), 5'-nucleotidase (E.C 3.1.3.5) and acetylcholinesterase (AChE, E.C. 3.1.1.7) activities in brain from adult rats. NTPDase and 5'-nucleotidase were measured in cortical synaptosomal fraction and AChE was measured in the homogenate of cerebral cortex and hippocampus. After the subchronic treatment (30 days), NTPDase activity was enhanced approximately 35% (p < 0.05) with ATP and ADP as substrates and no difference was observed in 5'-nucleotidase activity (AMP hydrolysis). In addition, AChE activity was enhanced in the cerebral cortex (22%, p < 0.05) and hippocampus (26%, p < 0.05) after the subchronic treatment. Mercury deposited in brain was measured by cold vapor (atomic absorption spectrometry) and no difference between the control and the subchronically treated group was observed. Here we showed for the first time that exposure to low levels of Hg2+, which resembles occupational exposure to low levels of mercury, caused a marked increase in NTPDase and AChE activities. The relationship of these alterations with the neurotoxicity of inorganic mercury deserves further studies.

5'-Nucleotidase↗

PubMed1994

Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase.

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

5'-Nucleotidase↗

PubMed1991

5'-Nucleotidase I from rabbit heart.

5'-Nucleotidase I (N-I) from rabbit heart was purified to homogeneity. After ammonium sulfate precipitation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose, AMP-agarose, and ADP-agarose. The pure enzyme has a specific activity of 318 mumol (mg of protein)-1 min-1. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate yields a subunit molecular weight of 40,000. N-I is activated by ADP but not by ATP, in contrast to the 5'-nucleotidase (N-II) purified by Itoh et al. (1986), which is activated by ATP and, less well, by ADP. N-I displays sigmoidal saturation kinetics in the absence of ADP and hyperbolic kinetics in the presence of ADP. Partially purified N-I was previously shown to prefer AMP over IMP as substrate (Truong et al., 1988); this has been confirmed for pure N-I. Comparison of AMP and ADP concentrations reported to occur in heart with the kinetic behavior of N-I implicates N-I as the enzyme responsible for producing adenosine under conditions leading to a rise in ADP and AMP, such as hypoxia or increased workload. N-I is not activated by the ADP analogue adenosine 5'-methylenediphosphonate (AOPCP) and is only weakly inhibited by relatively high concentrations of AOPCP, in contrast to 5'-nucleotidase from plasma membrane, which is powerfully inhibited by this analogue. N-I shows an absolute dependence on Mg2+ ions. Mn2+ and Co2+ ions can replace Mg2+ ions as activator; Ni2+ and Fe2+ are much less effective, while Ca2+, Ba2+, Zn2+, and Cu2+ fail to activate the enzyme.

5'-Nucleotidase↗

PubMed1975

Hydrolysis of ribonucleoside 3'-diphosphates by rye grass 3'-nucleotidase.

Rye grass 3'-nucleotidase has been purified to apparent homogeneity on Sephadex A-25 and CM-cellulose columns and shown to hydrolyze 2'-O-methyladenosine 3'-monophohate and 2'-deoxyadenosine 3'-monophosphate 35.8 and 542 times more slowly than the normal substrate (3'-AMP), verifying the importance of the 2'-beta-OH group of the substrate in the overall hydrolysis process. Although neither was hydrolyzed as rapidly as 3'-AMP, both the 2'-O-methyl and 2'-deoxy analogs acted as competitive inhibitors of the hydrolysis of 3'-AMP (K-m equals 0.12 mM), with apparent K-i's of 0.39 and 0.51 nM, respectively. In order to determine the possible susceptibility of naturally occurring ribonucleoside 3'-diphosphates, such as guanosine tetraphosphate (ppGpp), to 3'-phosphohydrolase activities, the 3'-nucleotidase was also employed in the attempted pyrophosphorolysis of adenosine 3'-diphosphate and guanosine tetraphosphate. Neither adenosine 3'-diphosphate nor guanosine tetraphosphate was degraded at a significant rate by the nucleotidase, relative to the normal substrate.

Adenine Nucleotides↗

PubMed2003

Influence of estrogen cytostatics on activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase.

We studied the effect of four conjugated synthetic derivatives of estrone and ethynylestradiol and bis-beta-chloroethylamine-containing substance on activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase. As differentiated from precursors, estrogen cytostatics decreased activity of plasma membrane enzymes. Reference preparations chlorophenacyl and estradiol had little effect on activity of 5'-nucleotidase and N+-K+-ATPase. These data suggest that damage to plasma membrane enzymes is related to the effect of estrogen cytostatic molecules. Test compounds produced an antiproliferative effect on estrogen-independent tumor cells, which strongly correlated with a decrease in activity of plasma membrane enzymes 5'-nucleotidase and N+-K+-ATPase. The derivative of ethynylestradiol with the cytostatic residue in the 3-position of the steroid nucleus (Po-714-11alpha) most significantly modulated enzyme activity.

5'-Nucleotidase↗

PubMed1969

Inhibition of 5'-nucleotidase from Ehrlich ascites-tumour cells by nucleoside triphosphates.

1. 5'-Nucleotidase activity was obtained in a soluble form after treatment of a particulate fraction from Ehrlich ascites-tumour cells with deoxycholate. The relative rates of hydrolysis of 6-thioinosine 5'-phosphate, UMP, AMP, CMP, GMP, IMP, xanthosine monophosphate, thymidine monophosphate and 2',3'-AMP were 180, 129, 100, 93, 83, 79, 46, 41 and 3 respectively. 2. Values found for the Michaelis constant were: AMP, 67+/-12mum; IMP, 111+/-8mum; GMP, 93mum. 3. ATP and thymidine triphosphate were competitive inhibitors of AMP hydrolysis (inhibitor constants 0.4 and 4.8mum respectively); UTP, GTP and CTP were mixed competitive and non-competitive inhibitors. Thymidine triphosphate was a competitive inhibitor of IMP hydrolysis (inhibitor constant 14.4mum) and ATP, UTP and GTP showed mixed competitive and non-competitive inhibition. 4. ATP, thymidine triphosphate, UTP, GTP and CTP did not completely inhibit hydrolysis of AMP, IMP and UMP; the concentrations of ATP required to inhibit AMP and IMP hydrolysis by 50% were 12 and 230mum respectively. 5. Non-hyperbolic curves relating activity to UMP concentration were obtained in the presence and absence of triphosphates. 6. After fractionation on Sephadex G-200 columns a single peak of 5'-nucleotidase activity (particle weight 120000-125000) was obtained with AMP, IMP and GMP as substrates. UMP hydrolysis was catalysed by enzyme in this peak and in two slower peaks corresponding to apparent particle weights of 32000 and 16000; a single component (particle weight 120000), reacting with UMP and insensitive to UTP inhibition, was obtained when the column was eluted with buffer containing 1mm-UMP. 7. The possible significance of the results in the regulation of tumour-cell 5'-nucleotidase is discussed.

Adenine Nucleotides↗

PubMed1980

Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.

Adenosine Deaminase↗