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)

For the determination of 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5;5'-Nase) in rat liver, a radiochemical double-labelling assay was developed. [14C]-labelled AMP which is hydrolyzed to [14C]-adenosine by 5'-Nase activity is added to crude liver homogenates. After 30 min, the process is stopped and [2-3H]-adenosine added to estimate the loss of [14C]-adenosine during separation by ion exchange column chromatography. The enzymatic reaction was found to be linear in correlation with the enzyme content and the incubation time. The specificity of the reaction was evaluated by addition of beta-glycerophosphate which acts as a competitive inhibitor to eliminate the catalytic effect of non-specific phosphatases, and addition of alpha, beta-methylene adenosine 5'-diphosphate, a specific inhibitor of 5'-Nase; both cause an almost complete suppression of enzyme activity.
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PMID:A double-labelling radioassay for the determination of 5'-nucleotidase activity. 45 39

Perfused rat hearts catalyze the hydrolysis of AMP added to the perfusion fluid at a rate of 35 mumol/g dry weight/min. The activity is specific for 5'-nucleoside monophosphates, little activity being observed with 2' and 3'-AMP. The enzyme exhibits Michaelis-Menten kinetics in situ and is inhibited competitively by adenosine-5'-alpha, beta-methylene diphosphonate (Ki = 13 muM). This, as well as the nucleotide specificity, confirms that the hydrolysis is catalyzed by 5'-nucleotidase. The maximum activity of 5'-nucleotidase in perfused hearts is equal to or greater than that found in heart homogenates; thus, all of the enzyme is accessible to AMP added externally. Hydrolysis of endogenous AMP was studied in the perfused heart. Under aerobic conditions hearts contain very low amounts of purine nucleosides, and little or no nucleoside is found in the effluent perfusate. Under anaerobic conditions hearts accumulate adenosine, inosine, and hypoxanthine and release all three substances into the perfusate. Hydrolysis of externally added AMP was also observed in perfused skeletal muscle and liver, at rates of 10 and 17 mumol/g dry weight/min, respectively.
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PMID:Studies of 5'-nucleotidase in the perfused rat heart. Including measurements of the enzyme in perfused skeletal muscle and liver. 97 76

In this study we report that preincubation of Dictyostelium discoideum membrane-bound adenylate cyclase with ATP over the concentration range 0.5 to 100 mM results in a loss of catalytic activity and that this effect persists even after removal of ATP. An analysis of the time course of this effect shows that, at 25 mM ATP, a 5- to 10-min preincubation results in 50% loss of activity. Additional studies on this effect showed that anhydride bond cleavage of ATP occurs during the preincubation. However, loss of catalytic activity is not porduced by ADP, AMP, cAMP, adenosine, pyrophosphate, or phosphate either separately or in pairs. Further, using the structural analogs adenosine 5'-(alpha, beta-methylene)triphosphate and adenyl-5'-yl imidodiphosphonate, we show that there is a direct correlation between alpha-beta-phosphoanhydride bond cleavage and the loss of catalytic activity. These results can be interpreted in terms of two classes of reaction mechanisms: either those involving covalent modifications or those involving a ligand-induced slow conversion of the adenylate cyclase from an active to an inactive form. Additional studies show that the addition of AMP to the reaction mixture, as well as removal of the membrane-bound 5'-nucleotidase activity, can prevent the loss of cyclase activity. These results suggest not only that adenylate cyclase activity is related to the AMP:ATP ratio but that the cyclase activity can be modified by the level of 5'-nucleotidase activity. Studies on the duration of the loss of activity produced by ATP show that following removal of ATP and additional incubation, a gradual recovery of cyclase activity is observed. This result suggests that under appropriate conditions the cyclase inactivation by ATP is reversible.
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PMID:Time-dependent changes in Dictyostelium discoideum adenylate cyclase activity upon incubation with ATP. 98 25

5'-Nucleotidase in perfused rat hearts, accessible to extracellular substrate [14C]-AMP contained in the perfusate, was compared to a partially purified 5'-nucleotidase from the same organ. Both activities were inhibited by ATP and, more effectively, by the ADP analog adenosine-alpha, beta-methylene diphosphate (APCP). The isolated enzyme showed a competitive inhibitor constant of 5.4 x 10(-8) M for APCP (Km of AMP = 1.4 x 10(-5) M). Although both activities were effectively inhibited by APCP, insufficient knowledge about the selectivity of this agent as a nucleotidase inhibitor does not permit a conclusion on whether or not the extracellular activity is identical to the partially purified enzyme.
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PMID:Inhibition of extracellular and purified 5'-nucleotidase from rat heart. 120 4

The regulation by ATP of Cl- secretion in T84 cells grown on filters was investigated by measuring short-circuit current (Isc = net Cl- secretion). ATP (greater than or equal to 10 microM) added to the basolateral side markedly stimulated Isc both in the presence and absence of forskolin-activated Isc. Fluorescence microscopy of cells loaded with the Ca2+ indicator fura-2 showed that ATP stimulated a transient increase in intracellular free Ca2+ concentration [Ca2+]i. The augmentation of forskolin-stimulated Isc by ATP was at least partly caused by mobilization of Ca2+ from an internal store because prior depletion of the store using ionomycin prevented the response. The activity sequence for stimulation of Isc in the presence of forskolin was adenosine 5'-O-(3-thiotriphosphate) = 5'-adenylylimidodiphosphate (AMP-PNP) greater than ATP greater than ADP greater than AMP, suggesting the presence of a P2 purinergic receptor. Neither beta, gamma-methyleneadenosine 5'-triphosphate nor alpha, beta-methyleneadenosine 5'-triphosphate increased the Isc. Stimulation of Isc by ATP in the absence of forskolin was at least partly due to the breakdown of ATP to AMP and adenosine, which act at P1 receptors to stimulate Isc, since 1) inhibition of the ecto-phosphohydrolase 5'-nucleotidase by alpha, beta-methylene-ADP partially inhibited stimulation of Isc by ATP, 2) the adenosine receptor antagonists caffeine and 8-phenyltheophylline markedly inhibited the ATP-stimulated Isc, and 3) AMP-PNP, a weakly hydrolyzable analogue of ATP, caused a much smaller increase in Isc compared with ATP. Adenosine had no effect on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purinergic receptor activation of Cl- secretion in T84 cells. 131 Feb 17

To test the hypothesis that 5'-nucleotidase activity during ischemia is attenuated by oxygen-derived free radicals, we measured ischemia-induced reactive hyperemic flow, adenosine release, and 5'-nucleotidase activity in dogs (n = 62). A 1-minute occlusion of the coronary artery caused reactive hyperemic flow (307 +/- 5 versus 92 +/- 1 ml.100 g-1.min-1 at baseline) with increased release of adenosine (14.4 +/- 1.4 versus 0.4 +/- 0.1 nmol.100 g-1.min-1 at baseline). Superoxide dismutase augmented (p less than 0.001) both peak coronary blood flow (333 +/- 6 ml.100 g-1.min-1) and repayment (436 +/- 12 versus 320 +/- 7 ml/100 g in the untreated group). Adenosine release during reperfusion was augmented (22.7 +/- 1.9 nmol.100 g-1.min-1, p less than 0.001), and 8-phenyltheophylline completely abolished the enhanced reactive hyperemia. Enzymatic assay of 5'-nucleotidase activity revealed that the administration of superoxide dismutase increases ecto-5'-nucleotidase activity in ischemic myocardium. When an inhibitor of ecto-5'-nucleotidase, alpha, beta-methyleneadenosine 5'-diphosphate, was administered, the effects of superoxide dismutase were completely abolished. Thus, we conclude that 1) the augmentation of reactive hyperemic flow caused by superoxide dismutase is attributed to the enhanced release of adenosine and 2) the enhanced release of adenosine over the untreated controls is attributed to the protection of ecto-5'-nucleotidase activity during ischemia.
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PMID:Superoxide dismutase enhances ischemia-induced reactive hyperemic flow and adenosine release in dogs. A role of 5'-nucleotidase activity. 149 5

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.
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PMID:Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine. 174 67

The effect of adenosine (ADO) on the recovery of cellular adenine nucleotides (AN) was evaluated in the cultured cells deprived of oxygen and substrates (ischemia) and in nonischemic cells (control). The primary cultured cells were obtained from microdissected rabbit proximal straight tubules. Ten-day-old cultured cells were made ischemic for 6 hr, and allowed to recover for 24 hr. At the end of ischemia, cells were incubated with ADO, theophylline (T), dipyridamole (D), coformycin (C) or combined agents for 3 hr. Total AN (TAN) were determined after 3 and 24 hr of recovery. The results, after 3 hr of incubation, suggest that in both control and ischemic cells, ADO is taken up by cultured cells and is preferentially converted to nucleotides. This effect is blocked by D, which inhibits ADO uptake, uninfluenced by C, which inhibits ADO deaminase and potentiated by T, which inhibits 5'-nucleotidase. After 24 hr of recovery, the beneficial effects of ADO alone or combined D, C, or T, on TAN were not seen in control cells. In contrast, in the ischemic cells, after 24 hr of recovery, ADO + T normalized ATP, ADP and TAN to the preischemic levels. T alone significantly increased ATP after 24 hr of recovery. To demonstrate further that the beneficial effect of T is due to inhibition of 5'-nucleotidase, cells were treated with adenosine alpha, beta-methylene diphosphate in the same manner as T. Combined ADO + adenosine alpha, beta-methylene diphosphate normalized ATP, ADP and TAN after 24 hr of recovery. This finding suggests that inhibition of 5'-nucleotidase improves postischemic AN.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Roles of adenosine and theophylline on the recovery of adenine nucleotides in postischemic cultured renal tubular cells. 203 18

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.
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PMID:Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides. 215 57

Acetylcholine and ATP are costored and coreleased during synaptic activity at the electric organ of Torpedo. It has been suggested that released ATP is converted to adenosine at the synaptic cleft, and in turn this nucleoside would depress the evoked release of acetylcholine. In the present communication we have used a chemiluminescent reaction that let us to monitor continuously the presence of adenosine in this preparation. The chemiluminescent reaction is based on the conversion of adenosine into uric acid and H2O2 by adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase enzymes. The hydrogen peroxide has been detected by peroxidase-luminol mixture. The reaction has a sensitivity on the picomol range and discerned between Adenosine, AMP, ADP, and ATP. We have developed this technique in the hope of understanding whether adenosine is released during synaptic activity or it comes from the released ATP. We have studied the release or formation of adenosine in fragments of the electric organ and in isolated cholinergic nerve terminals obtained from it. In both conditions we have followed the effect of potassium stimulation upon the detection of adenosine. Potassium stimulation increased the extracellular adenosine either in slices or the synaptosomal fraction of Torpedo electric organ. The presence of alpha, beta-methylene ADP, an inhibitor of 5'-nucleotidase, inhibits the detection of adenosine, suggesting that extracellular adenosine is a consequence of ectocellular dephosphorylation of released ATP.
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PMID:The release of adenosine at the electric organ of Torpedo. A study using a continuous chemiluminescent method. 232 27


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