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)

2',3'-Dideoxyinosine (ddlno) is a potent and selective inhibitor of human immunodeficiency virus in human lymphoid cells and monocytes/macrophages. Earlier studies [J. Biol. Chem. 263:15354 (1988)] showed that anabolism of ddlno in human lymphoid cells is mediated via an initial step of phosphorylation and subsequent amination to dideoxy-AMP via adenylosuccinate synthetase/lyase. Evidence was obtained that neither adenosine kinase nor deoxycytidine kinase is involved in the phosphorylation of this compound in human lymphoid cells. We now find that, in the presence of MgCl2, KCl, and inosine-5'-monophosphate as phosphate donor, purified cytosolic 5'-nucleotidase catalyzed the phosphorylation of ddlno. Although not phosphate donors, ATP, diadenosine tetraphosphate, and glycerate-2,3-bisphosphate stimulate this phosphorylation by the nucleotidase 4-5-fold. In addition to ddlno, the antiviral nucleoside analogs 2',3'-dideoxyguanosine and carbovir were substrates for this enzyme. The relative phosphorylation of these compounds varied with the concentration of the phosphate donor IMP. Approximate Km values of the nucleotidase for inosine, ddlno, dideoxyguanosine, and carbovir were, respectively, 3.4, 0.5, 0.9, and 1.7 mM. Although the substrate activity of dideoxynucleosides is inefficient, it appears likely that this nucleotidase is responsible for the metabolism of these compounds to their active nucleotides, yielding antiviral activity in human lymphoid cells.
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PMID:Phosphorylation of 2',3'-dideoxyinosine by cytosolic 5'-nucleotidase of human lymphoid cells. 254 85

Mature secretory granules in paraneurons contain ATP amongst other small messenger molecules. In the islet organ such stores of adenine nucleotides readily can be demonstrated by means of the quinacrine fluorescence method. ATP is co-released together with other granule constituents when the major hormones are exocytosed. The distribution of ATP splitting enzymic activities was studied in the pancreas of the mouse and rat, in order to obtain information on the possible fate of this small messenger molecule. ATPase, ADPase, and AMPase (5'-nucleotidase) were demonstrated with lead precipitation methods, L-tetramisole was used to inhibit unspecific alkaline phosphatase (alPase); alPase activities were shown with tetrazolium methods, using 5-bromo-4-chloro-3-indoxyl phosphate as substrate. Most endothelial cells of the vascular bed, both in the exocrine and in the endocrine pancreas, are reactive for ATPase, ADPase, AMPase and alPase. Smooth muscle cells are strongly reactive for ATPase and AMPase, vascular adventitial fibroblasts (veil cells) stain for ATPase and alPase, as do some lamellar cells at the islets surface. Staining for ADPase serves as a selective method to demonstrate the vascular bed. Comparable results are obtained with the alPase reaction, though insular non-B-cells are also reactive. ATPase staining is less useful for demonstrating vascular connections because moderate reactivity of exocrine parenchyma and adventitial tissue obscures the picture. AMPase activity is strong in the venous segments of the capillary net and in collecting veins but the reaction obviously does not demonstrate significant portions of the residual capillary network. Weak AMPase activity is seen in the insular parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Fate of ATP in secretory granules: phosphohydrolase studies in pancreatic vascular bed. 255 47

A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca2+ + Mg2+)ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate.
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PMID:A microtiter plate assay for inorganic phosphate. 255 7

Adenosine may modulate blood flow and electrical activity in heart in response to changes in myocardial energy metabolism. In the present study, 31P NMR spectroscopy was used to examine the relation between cytosolic phosphate metabolite levels and release of adenosine into the venous effluent of isovolumic heart during graded low-flow ischaemia or metabolic stimulation with isoproterenol. When coronary flow rate was varied in steps between 1.6 and 12 ml/min/g, cytosolic ATP levels did not change significantly but the phosphorylation potential exhibited a linear correlation with flow rate below approximately 7 ml/min/g. Purine release (adenosine and inosine) correlated linearly with the cytosolic phosphorylation potential and free AMP concentration. Metabolic stimulation of hearts with isoproterenol (0.4, 3.0, and 60 nM), produced a significant fall in cytosolic ATP levels and decreased the cytosolic phosphorylation potential. Purine release in these hearts increased exponentially as the cytosolic phosphorylation potential dropped, and as cytosolic free AMP increased. These results support a link between the phosphorylation potential and the mechanism of adenosine production during ischaemia and metabolic stimulation. Presumably, this link is the activity of the enzyme 5'-nucleotidase, which is responsible for converting AMP to adenosine, together with the concentration of its substrate, AMP. In low-flow ischaemia, cytosolic AMP may control adenosine formation. With isoproterenol stimulation, a more complex relationship exists, indicating possible allosteric regulation of the enzyme(s) responsible for adenosine formation, in addition to changes in AMP concentration.
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PMID:Adenosine production and energy metabolism in ischaemic and metabolically stimulated rat heart. 255 22

Quantitative assessment of high-energy phosphate levels, including degradation or utilization during ischemia, has not previously been performed in infants and children. Animal experiments suggest that high-energy phosphate metabolism varies with maturation. To help answer these questions, 24 patients aged 2 months to 8 years underwent myocardial biopsy immediately after the institution of cardiopulmonary bypass (16 to 20 degrees C). Additional samples were obtained at 16 and 45 minutes after aortic cross-clamping and administration of cardioplegia (St. Thomas's solution) (in vivo ischemia). Seven patients also underwent major myocardial resection. Resected specimens were placed in a 37 degrees C bath and divided into equal-sized samples that were removed at ten-minute intervals (in vitro ischemia). All samples were immersed in liquid nitrogen and analyzed for adenine nucleotide pool metabolites using high-performance liquid chromatography. Levels of adenosine triphosphate were high before cross-clamping but diminished during the period of protected ischemia. Adenosine triphosphate loss was much more pronounced in patients less than 18 months old (p less than 0.05) and was associated with accumulation of adenosine monophosphate and inosine, a finding not seen in patients more than 18 months old (p less than 0.05). The same trends documented during in vivo ischemia were noted during in vitro ischemia. Immaturity of 5'-nucleotidase results in accumulation of adenosine monophosphate during ischemia. It is known that 5'-nucleotidase is present in neonatal myocardial cell membranes and absent from the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Myocardial adenine nucleotide metabolism in pediatric patients during hypothermic cardioplegic arrest and normothermic ischemia. 273 Jan 89

Growth of cells of the potentially zoopathogenic fungus Basidiobolus haptosporus on a nutritionally defined medium with xanthine or urate as the nitrogen source results in greatly increased populations of microbodies. Modified Gomori procedures at the electron microscopic level suggested the single limiting membrane (and in some cases the granular matrix) of immature microbodies to be the exclusive subcellular locale(s) of alkaline phosphatase, 5'-nucleotidase and nucleoside diphosphatase activities. When grown in the presence of low inorganic phosphate, additional alkaline phosphatase activity was further identified cytochemically at and along profiles of endoplasmic reticulum and on inclusions previously described as "double-membraned vesicles". Cytochemical localization of acid phosphatase at microbody membranes was minimal if not ambiguous; Mg++-dependent adenosine triphosphatase and glucose-6-phosphatase were not identified at these locales. Quantitative biochemical estimates of alkaline phosphatase activity levels in particulate fractions initially increased with age of cells, perhaps as a function of the cultural induction and marked increase in immature microbody populations. We suggest that this enzyme may participate in some manner with protein translocation mechanisms associated with microbody biogenesis, ontogeny, and/or physiological function.
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PMID:Electron cytochemical demonstration of phosphatase activity with microbody membranes of Basidiobolus haptosporus. 282 62

The distribution of hepatic binding sites for the calcium-mobilizing second messenger, inositol 1,4,5-trisphosphate (IP3), was analyzed in subcellular fractions of the rat liver by binding studies with [32P]IP3 and compared with the Ca2+ release elicited by IP3 in each fraction. Three major subcellular fractions enriched in plasma membrane, mitochondria, and endoplasmic reticulum were characterized for their 5'-nucleotidase, glucose-6-phosphatase, succinate reductase, and angiotensin II binding activities. The fraction enriched in plasma membrane showed 7- and 20-fold increases in IP3 binding capacity over those enriched in endoplasmic reticulum and mitochondria, respectively, and contained a single class of high-affinity binding sites with Kd of 1.7 +/- 1.0 nM and concentration of 239 +/- 91 fmol/mg protein. IP3 binding reached equilibrium in 30 min at 0 degrees C, and the half-time of dissociation was about 15 min. The specificity of the IP3 binding sites was indicated by their markedly lower affinities for inositol 1-phosphate, phytic acid, fructose 1,6-bisphosphate, 2,3-bisphosphoglycerate, and inositol 1,3,4,5-tetrakisphosphate. The Ca2+-releasing activity of IP3 in the subcellular fractions was monitored with the fluorescent indicator, Fura-2. All three fractions showed ATP-dependent Ca2+ uptake and rapidly released Ca2+ in response in IP3. The fraction enriched in plasma membrane was the most active in this regard, releasing 174 +/- 67 pmol Ca2+/mg of protein compared to 45 +/- 10 and 48 +/- 7 pmol/mg protein for the fractions enriched in endoplasmic reticulum and mitochondria, respectively. These data suggest that the [32P]IP3 binding sites represent specific intracellular receptors through which IP3 mobilizes Ca2+ from a storage site associated (or co-purifying) with the plasma membrane of the rat liver. It is likely that a specialized vesicular system (to which IP3 can bind and trigger the release of Ca2+) is located in close proximity with the plasma membrane and is thus adjacent to the site at which IP3 is produced during stimulation of the hepatocyte by Ca2+-mobilizing hormones.
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PMID:Characterization of inositol 1,4,5-trisphosphate receptors and calcium mobilization in a hepatic plasma membrane fraction. 283 98

We have used both the enzyme cytochemical method with lead nitrate as a capture agent and an immunological method at the electron microscope level to localize plasma membrane 5'-nucleotidase in rat peritoneal resident macrophages during the initial interactions of latex beads or heat-killed Escherichia coli with the cell during phagocytosis. In macrophages at rest, cytochemical reaction product was evenly distributed along the external surface of the plasma membrane. However, when the cells were phagocytosing latex beads or bacteria, reaction product covered the entire surface of the adhering particles. To determine whether the apparent redistribution of 5'-nucleotidase onto the adhering particle was fact or artifact, we localized 5'-nucleotidase using a monoclonal antibody and an immunogold labelling technique. In macrophages binding or beginning to ingest bacteria, gold particles were distributed along the plasma membrane, except at the sites of cell-bacterium internalization. More significantly, the adhering bacteria were free of gold particles and therefore had no 5'-nucleotidase on their surfaces. Latex beads proved to be unsuitable as a test particle because the gold particles stuck to them non-specifically. We conclude that the artifactual redistribution of lead-phosphate reaction product is a major drawback of enzyme cytochemical methods when used on cell surfaces and that the immunogold labelling technique is more reliable.
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PMID:Pitfalls in ecto-5'-nucleotidase enzyme cytochemistry as demonstrated by the immunogold-labelling technique on macrophages. 283 34

1. A 5'-nucleotidase with a strong preference for AMP over IMP was characterized in homogenates and subcellular fractions of pigeon heart by using concentrations of ATP, ADP and AMP which mimicked those present in the ischaemic tissue. 2. The AMP-5'-nucleotidase had a neutral pH optimum and an apparent Km in the range 4.6-5.2 mM. It was stimulated by ATP plus ADP, and was inhibited by other nucleoside monophosphates, Pi and p-nitrophenyl phosphate, but not by ribose 5-phosphate or beta-glycerophosphate. The enzyme was not inhibited by [alpha beta-methylene] ADP or by 5'-deoxy-5'-isobutylthioadenosine, an inhibitor of the previously purified IMP-preferring cytosolic 5'-nucleotidase. 3. Subcellular-fractionation studies indicated that the enzyme has access to cytosolic AMP, although it may be associated by weak ionic interactions with an organelle present in the low-speed particulate fraction. 4. A 5'-nucleotidase was detected under similar conditions in homogenates of rat heart. 5. The activity of the pigeon heart AMP-5'-nucleotidase was sufficient to account for previously measured rates of ischaemia-induced adenosine formation. The similar activity in rat heart could, however, account for only part of ischaemia-induced adenosine formation in this tissue.
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PMID:The pigeon heart 5'-nucleotidase responsible for ischaemia-induced adenosine formation. 284 63

A human placental soluble "high Km" 5'-nucleotidase has been separated from "low Km" 5'-nucleotidase and nonspecific phosphatase by AMP-Sepharose affinity chromatography. The enzyme was purified 8000-fold to a specific activity of 25.6 mumol/min/mg. The subunit molecular mass is 53 kDa, and the native molecular mass is 210 kDa, suggesting a tetrameric structure. Soluble high Km 5'-nucleotidase is most active with IMP and GMP and their deoxy derivatives. IMP is hydrolyzed 15 times faster than AMP. The enzyme has a virtually absolute requirement for magnesium ions and is regulated by them. Purine nucleoside 5'-triphosphates strongly activate the enzyme with the potency order dATP greater than ATP greater than GTP. 2,3-Diphosphoglycerate activates the enzyme as potently as ATP. Three millimolar ATP decreased the Km for IMP from 0.33 to 0.09 mM and increased the Vmax 12-fold. ATP activation was modified by the IMP concentration. At 20 microM IMP the ATP-dependent activation curve was sigmoidal, while at 2 mM IMP it was hyperbolic. The A0.5 values for ATP were 2.26 and 0.70 mM, and the relative maximal velocities were 32.9 and 126.0 nmol/min, respectively. Inorganic phosphate shifts the hyperbolic substrate velocity relationship for IMP to a sigmoidal one. With physiological concentrations of cofactors (3 mM ATP, 1-4 mM Pi, 150 mM KCl) at pH 7.4, the enzyme is 25-35 times more active toward 100 microM IMP than 100 microM AMP. These data show that: (a) soluble human placental high Km 5'-nucleotidase coexists in human placenta with the low Km enzyme; (b) under physiological conditions the enzyme favors the hydrolysis of IMP and is critically regulated by IMP, ATP, and Pi levels; and (c) kinetic properties of ATP and IMP are each modified by the other compound suggesting complex interaction of the associated binding sites.
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PMID:High Km soluble 5'-nucleotidase from human placenta. Properties and allosteric regulation by IMP and ATP. 284 5


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