Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We demonstrate that a mucoid, alginate-producing strain of Pseudomonas aeruginosa isolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5'-nucleotidase, and ATP-modifying enzymatic activities. The secretion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presence of 5.0 mM Ca2+ or Mg2+. Interestingly, the secretion is triggered primarily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1. The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracellular Ndk and demonstrates similar enzymatic properties. The N-terminal sequence of the purified ATPase isolated from an ndk knockout mutant shows its identity with that of the heat shock chaperonin Hsp60. During fractionation, the flowthrough fraction from a Mono Q column demonstrates the presence of 5'-nucleotidase, adenylate kinase, and a putative ATP reductase activity. These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP or inhibited by pretreatment of macrophages with oxidized ATP (oATP). The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane. Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death. Thus mucoid P. aeruginosa cells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation. Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.
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PMID:P2Z-Independent and P2Z receptor-mediated macrophage killing by Pseudomonas aeruginosa isolated from cystic fibrosis patients. 1049

In Scrobicularia plana testis, a nuclear acid phosphatase (ACPase) activity was detected in mid and late spermatids with the improved Gomori-chloride procedure. Lead deposits were first observed in mid spermatids at focal points over condensed chromatin strands, increasing in density as chromatin further condensated. In late spermiogenesis, lead deposits became concentrated between chromatin aggregates, and after total DNA compaction were transfered to the nuclear periphery and then shed into the cytoplasm. The specificity of the nuclear ACPase was tested against different pH values (3.9, 7.2, 7.8, 9.0), substrates (TPP, IDP, TMP, p-NCS, ATP, GTP, AMP, ADP, AMP-PNP) and inhibitors (NaF, levamisole, Zn, vanadate, theophylline). To further specify the nature of this nuclear ACPase, other enzymes were comparatively studied at their optimal pH values and at pH 5.0: nucleoside-diphosphatase, thiamin-pyrophosphatase, inorganic trimetaphosphatase, lysosomal arylsulfatases A and B, ATPase, GTPase, 5'-nucleotidase, adenylate kinase, and adenylate cyclase. Several other controls were introduced to exclude artefactual deposits induced by lead ions and tissue molecules. The results showed that the enzyme has an optimal pH at 5.0, a high specific affinity for beta-GP, and is inhibited by NaF, which suggests that it behaves as a type B-ACPase, and all controls demonstrated the specificity of the enzymic activity. Because lead deposits were specifically and temporally associated with spermatid chromatin condensation, when DNA and RNA synthesis, histones, phosphoproteins and RNA molecules strongly decrease, it is possible to suggest that the nuclear ACPase could be associated with DNA processing during chromatin compaction or involved in the hydrolysis of 2' and 3' nucleotides resulting from nuclear RNase action during RNA degradation.
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PMID:Chromatin condensation during Scrobicularia plana spermiogenesis: a controlled and comparative enzymatic ultracytochemical study. 1079 22

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5'-nucleotidase activity, the level of secretion of the 5'-nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as alpha2-macroglobulin. As macrophage-or mast cell surface-associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP-using enzymes generate various phosphorylated or non-phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.
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PMID:Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells. 1093 Dec 97

A nonmucoid clinical isolate of Pseudomonas aeruginosa, strain 808, elaborated ATP-dependent and ATP-independent types of cytotoxic factors in the growth medium. These cytotoxic factors, active against macrophages, were secreted during the exponential phase of growth in a complex medium. Commensurate with the appearance of the cytotoxic activities in the cell-free growth medium, several ATP-utilizing enzymic activities, such as adenylate kinase, nucleoside diphosphate kinase and 5'-nucleotidase (ATPase and/or phosphatase), were detected in the medium. These ATP-utilizing enzymes are believed to convert external ATP, presumably effluxed from macrophages, to various adenine nucleotides, which then activate purinergic receptors such as P2Z, leading to enhanced macrophage cell death. Pretreatment of macrophages with periodate-oxidized ATP (oATP), which is an irreversible inhibitor of P2Z receptor activation, prevented subsequent ATP-induced macrophage cell death. A second type of cytotoxic factor(s) operated in an ATP-independent manner such that it triggered activation of apoptotic processes in macrophages, leading to proteolytic conversion of procaspase-3 to active caspase-3. This cytotoxic factor(s) did not appear to act on procaspase-3 present in macrophage cytosolic extracts. Intact macrophages, when exposed to the cytotoxic factor(s) for 6-16 h, underwent apoptosis and demonstrated the presence of active caspase-3 in their cytosolic extracts. Interestingly, two redox proteins, azurin and cytochrome c(551), were detected in the cytotoxic preparation. When cell-line-derived or peritoneal macrophages or mast cells were incubated overnight with Q-Sepharose column flow-through fraction or with a mixture of azurin and cytochrome c(551), they underwent extensive cell death due to induction of apoptosis.
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PMID:Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependent, P2Z-receptor-mediated necrosis and ATP-independent, caspase-mediated apoptosis. 1102 27

Cell surface ecto-nucleotidases are considered the major effector system for inactivation of extracellular adenine nucleotides, whereas the alternative possibility of ATP synthesis has received little attention. Using a TLC assay, we investigated the main exchange activities of 3H-labeled adenine nucleotides on the cultured human umbilical vein endothelial cells. Stepwise nucleotide degradation to adenosine occurred when a particular nucleotide was present alone, whereas combined cell treatment with ATP and either [3H]AMP or [3H]ADP caused unexpected phosphorylation of 3H-nucleotides via the backward reactions AMP --> ADP --> ATP. The following two groups of nucleotide-converting ecto-enzymes were identified based on inhibition and substrate specificity studies: 1) ecto-nucleotidases, ATP-diphosphohydrolase, and 5'-nucleotidase; 2) ecto-nucleotide kinases, adenylate kinase, and nucleoside diphosphate kinase. Ecto-nucleoside diphosphate kinase possessed the highest activity, as revealed by comparative kinetic analysis, and was capable of using both adenine and nonadenine nucleotides as phosphate donors and acceptors. The transphosphorylation mechanism was confirmed by direct transfer of the gamma-phosphate from [gamma-32P]ATP to AMP or nucleoside diphosphates and by measurement of extracellular ATP synthesis using luciferin-luciferase luminometry. The data demonstrate the coexistence of opposite, ATP-consuming and ATP-generating, pathways on the cell surface and provide a novel mechanism for regulating the duration and magnitude of purinergic signaling in the vasculature.
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PMID:Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions. 1114 13

Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5'-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with (3)H-labelled nucleotides and adenosine as substrates, direct evaluation of gamma-phosphate transfer from [gamma-(32)P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20 kDa surface protein was observed following incubation of Namalwa B cells with [gamma-(32)P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.
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PMID:The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells. 1209 90

Extracellular ATP and other purines play a crucial role in the vasculature, and their turnover is selectively governed by a network of ectoenzymes expressed both on endothelial and hematopoietic cells. By studying the whole pattern of purine metabolism in human serum, we revealed the existence of soluble enzymes capable of both inactivating and transphosphorylating circulating purines. Evidence for this was obtained by using independent assays, including chromatographic analyses with 3H-labeled and unlabeled nucleotides and adenosine, direct transfer of gamma-terminal phosphate from [gamma-32P]ATP to NDP/AMP, and bioluminescent measurement of ATP metabolism. Based on substrate-specificity and competitive studies, we identified three purine-inactivating enzymes in human serum, nucleotide pyrophosphatase (EC 3.6.1.9), 5'-nucleotidase (EC 3.1.3.5), and adenosine deaminase (EC 3.5.4.4), whereas an opposite ATP-generating pathway is represented by adenylate kinase (EC 2.7.4.3) and NDP kinase (EC 2.7.4.6). Comparative kinetic analysis revealed that the Vmax values for soluble nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent Km values for serum enzymes were fairly comparable and varied within a range of 40-70 micro mol/l. Identification of soluble enzymes contributing, along with membrane-bound ectoenzymes, to the active cycling between circulating ATP and other purines provides a novel insight into the regulatory mechanisms of purine homeostasis in the blood.
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PMID:Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions. 1275 41

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5'-nucleotidase (5'-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5'-NT, and PNP in tumor tissue than in neighboring mucosa (p > 0.01 for ADA, AK, and PNP; p > 0.05 for 5'-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5'-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.
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PMID:Enzyme activities controlling adenosine levels in normal and neoplastic tissues. 1529 91

Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N(6)-(delta(2)-isopentenyl)adenosine. Electrophoretic analysis indicated that only N(6)-(delta(2)-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5'-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine.The phosphorylation of the cytokinin and adenosine required ATP and Mg(2+). The pH optimum was from 6.8 to 7.2 for both the cytokinin and adenosine. At pH 7 and 37 C the Km and V(max) for the cytokinin were 31 mum and 8.3 nmoles per mg protein per minute, and the values for adenosine were 8.7 mum and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ phosphorylated N(6)-(delta(2)-isopentenyl)adenosine. These preparations also phosphorylated N(6)-(delta(2)-isopentenyl)adenine when 5-phosphorylribose-1-pyrophosphate was present.
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PMID:Phosphorylation of cytokinin by adenosine kinase from wheat germ. 1665 70

In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5'-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for divalent cations (Mg(2+)>Ca(2+)). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K(m) values were 464.2+/-86.6 microM (mean+/-SEM, n=4), 137.0+/-31 microM (mean+/-SEM, n=5) and 44.8+/-10.2 microM (mean+/-SEM, n=4), and V(max) values were 655.0+/-94.6 (mean+/-SEM, n=4), 236.3+/-27.2 (mean+/-SEM, n=5) and 177.6+/-13.8 (mean+/-SEM, n=5) nmol of inorganic phosphate min(-1) mg of protein(-1) for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5'-nucleotidase. The presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth.
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PMID:Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: molecular identification, kinetic characterization and biochemical properties. 1716 79


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