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)

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase has been purified from calf thymus. Since the same protein is able to catalyze both the hydrolysis and the interconversion of several nucleoside monophosphates, it is necessary to study the effect of different metabolites and assay conditions on both activities in order to elucidate their physiological roles. We describe herein a method which allowed us to follow both activities contemporaneously in the same assay mixture. The method takes advantage of the observation that deoxyGMP is both a good substrate for hydrolysis and a good phosphate donor for the phosphotransferase reaction, but its dephosphorylated product, deoxyguanosine, is not a phosphate acceptor. As a consequence, it is possible to follow both the deoxyguanosine production and the transfer of phosphate from deoxyGMP to the best phosphate acceptor, inosine, during the reaction, applying a method for the chromatographic separation on HPLC of both substrates (inosine and deoxyGMP) and both products (IMP and deoxyguanosine). The method was applied to the determination of the KM for inosine.
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PMID:Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a single assay for a bifunctional enzyme. 830 94

The antiviral activity of the purine dideoxynucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxyinosine (ddI) is dependent on their conversion into ddA triphosphate in vivo. 5-Amino-4-imidazolecarboxamide riboside (AICA riboside), a natural metabolite in purine biosynthetic pathways, is converted into IMP, a substrate for the biosynthesis of adenine and guanine nucleotides, and enhances the intracellular purine nucleotide pools. Because IMP also serves as a phosphate donor in the anabolic phosphorylation of ddI (and ddA) into ddI monophosphate by the cytosolic enzyme 5'-nucleotidase, we investigated the effects of AICA riboside on the phosphorylation and antiretroviral activity of these purine nucleoside analogs. At an AICA riboside concentration of 0.5 mM, there was a approximately 2-fold increase in the intracellular ATP and GTP levels, whereas a nearly 8-fold increase was observed for the phosphorylation of ddA (or ddI). A marked reduction in intracellular pools of the pyrimidine nucleotides CTP and UTP was observed in AICA riboside-treated cells and inhibited cell proliferation. However, this growth inhibition was prevented by the addition of uridine to the cultures. Cells pretreated with AICA riboside and ddI were less susceptible to human immunodeficiency virus (HIV) infection and synthesized reduced levels of HIV proviral DNA. A 10-fold potentiation of the effectiveness of ddI against both wild-type HIV (HIVIIIB) and a ddI-resistant variant HIV was observed in the presence of 0.5 mM AICA riboside. These results show that AICA riboside modulates the anabolism and antiviral activity of ddI, and they have implications for possible therapies with dideoxynucleosides.
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PMID:5-Amino-4-imidazolecarboxamide riboside potentiates the metabolism and anti-human immunodeficiency virus activity of 2',3'-dideoxyinosine. 834 Dec 76

During induced ischemia for cardiac surgery, 5'-nucleotidase (5NT) catalyzes nucleotide breakdown by dephosphorylating AMP and IMP to diffusible precursors--adenosine and inosine. These precursors become unavailable upon reperfusion washout limiting nucleotide resynthesis, resulting in poor postischemic function. Neonatal hearts, which are more resistant to ischemia than adults, have low 5NT activity, trapping available precursors. Adult rabbit hearts given cardioplegia with a 5NT inhibitor, pentoxifylline, demonstrated improved postischemic contractility, compliance, and myocardial oxygen consumption after 120 min of 34 degrees C ischemia. To determine if this improved function was a result of enhanced nucleotide precursor availability during or following ischemia, total nondiffusible nucleotides, ATP, ADP, AMP, and IMP, and total diffusible nucleotides, adenosine, inosine, hypoxanthine, and xanthine, were measured by HPLC at end ischemia, 1 and 15 min after reperfusion. While all preischemic values were equivalent, pentoxifylline-treated hearts had significantly greater total non-diffusible nucleotides at end ischemia, 1 and 15 min after reperfusion. Additionally, pentoxifylline-treated hearts had significantly greater total diffusible nucleosides at end ischemia and 1 min after reperfusion, but were equal to control at 15 min after reperfusion. Furthermore, coronary sinus effluent had a significantly higher release of total diffusible nucleosides in control vs pentoxifylline-treated hearts. The data indicate that precursor trapping with pentoxifylline prevented nucleotide catabolism to diffusible precursors and enhanced postischemic nucleotide availability. We postulate the increased precursor availability augmented myocardial nucleotide resynthesis and correlated with the improved functional recovery noted. This strategy may have application in adult cardiac surgery.
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PMID:Precursor trapping: a "neonatal" mechanism of myocardial protection. 841 63

Soluble low Km 5'-nucleotidases have been purified from human cultured T- and B-lymphoblasts to compare their properties and to examine the mechanism of different rates of nucleotide dephosphorylation. The enzyme from B-lymphoblasts (MGL-8) was 4385-fold purified with a specific activity of 114 mumol/min/mg, while the enzyme from T-lymphoblasts (CEM, MOLT-4) was 4355-fold purified with a specific activity of 35 mumol/min/mg. The activity of both enzymes have an absolute requirement for Mg++. The B-cell enzyme has maximum activity with Mg2+ > Mn2+ > Co2+, while the T-cell enzyme had maximum activity with Co2+ > Mn2+ > Mg2+. The optimum activity was at pH 7.4-9.0 for the B-cell enzyme and pH 9.0 for the T-cell enzyme. Substrate specificity was the same for both enzymes with the following relative Vmax values: CMP > UMP > dUMP > dCMP > dAMP > IMP > GMP > dIMP > dGMP. The Km values for AMP and IMP were 12 and 25 microM for the B-cell enzyme, and 7.0 and 12 microM for the T-cell enzyme. ATP and ADP are competitive inhibitors of these enzymes with apparent Ki values of 100 and 20 microM for the B-cell enzyme, and 44 microM and 8 microM for the T-cell enzyme, respectively. The apparent molecular mass by gel filtration column chromatography is 145 kD for the B-cell enzyme and 72 kDa for the T-cell enzyme. The subunit molecular masses by Western blots are 69.2 kD for both enzymes. These properties suggest that the B-lymphoblast enzyme is identical or similar to the enzyme from human placenta. However, the T-cell enzyme has some different properties. We conclude that these differences plus a lower content of low Km 5'-nucleotidase in T-cells may account for the decreased ability of T-lymphoblasts to dephosphorylate nucleotides and may contribute to the selective cytotoxicity of deoxyribonucleosides for T-lymphoblasts as compared to B-lymphoblasts.
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PMID:Altered properties of human T-lymphoblast soluble low Km 5'-nucleotidase: comparison with B-lymphoblast enzyme. 845 Jun 71

The elevation of adenosine levels induced by anoxia in isolated rat hepatocytes has been shown to result mainly from an arrest of the recycling of the nucleoside by adenosine kinase [Bontemps, Vincent and Van den Berghe (1993) Biochem. J. 290, 671-677]. To assess the activity of the latter enzyme in intact hepatocytes, incorporation of radioactive adenosine into the cells' adenine nucleotides was measured. Unexpectedly, despite the near-absence of ATP in anoxic cells, 40% of 50 microM [8-14C]adenosine was still incorporated into adenylates over 5 min. Moreover, whereas unlabelled and labelled adenosine were utilized in parallel in normoxic cells, uptake of [8-14C]adenosine did not correspond to a net disappearance of adenosine in anoxic cells. Addition of 1 mM unlabelled adenosine to anoxic hepatocytes in which the adenine nucleotides had been prelabelled with [U-14C]adenine induced an immediate loss of their radioactivity. The latter was recovered in the form of adenosine, but the size of the adenylate pool was not modified. Taken together, these results suggest the occurrence of an exchange reaction between AMP and adenosine. Incubation of Sephadex G-25-filtered high-speed supernatants of rat liver with 20 microM [8-14C]adenosine, 10 mM MgCl2 and 1 mM AMP resulted in the labelling of AMP in the total absence of ATP. This labelling was influenced by effectors of both adenosine kinase and cytosolic IMP-GMP 5'-nucleotidase; the latter is known to catalyse an exchange reaction [Worku and Newby (1982) Biochem. J. 205, 503-510]. Chromatography of cytosolic fractions of rat liver on DEAE-Sepharose, followed by Sephacryl S-200 and AMP-Sepharose, demonstrated that the exchange reaction between adenosine and AMP co-purified with adenosine kinase. It is concluded that incorporation of labelled adenosine into adenine nucleotides should not be considered to be proof of adenosine kinase activity in anoxia.
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PMID:Phosphorylation of adenosine in anoxic hepatocytes by an exchange reaction catalysed by adenosine kinase. 845 94

1. Studies in rat polymorphonuclear leucocytes have suggested that 5'-deoxy-5'-isobutylthioadenosine (IBTA), an inhibitor of the IMP-selective cytosolic 5'-nucleotidase, may be used to test its role in adenosine formation in intact cells. We investigated adenosine formation in neonatal and adult rat cardiomyocytes. 2. 2-Deoxyglucose (30 mM) with oligomycin (2 micrograms/ml) induced a 90-100% fall in ATP concentration in 10 min in neonatal and 60 min in adult heart cells. Adenosine accumulation was substantially increased, accounting for 13% of the fall in ATP concentration in neonatal cells and 56% in adult cells. 3. Anti-(rat liver ecto-5'-nucleotidase) serum did not inhibit adenosine accumulation. Furthermore, dipyridamole (10 microM), a nucleoside-transport blocker, inhibited by 80% the appearance of the newly formed adenosine in the medium, showing that adenosine is produced intracellularly by both adult and neonatal-rat myocytes in response to inhibition of oxidative metabolism. 4. IBTA (3 mM) inhibited by 80% the appearance of adenosine in the medium, but did not inhibit total adenosine accumulation by neonatal-rat myocytes and only modestly inhibited total adenosine accumulation by adult myocytes. 5. IBTA, like dipyridamole, inhibited incorporation of extracellular adenosine (10 microM) into neonatal and adult ventricular myocyte nucleotides by 60-70%. Transport of IBTA (100 microM) into the cells did not appear to be inhibited by dipyridamole (30 microM). 6. We conclude that IBTA acted primarily to inhibit adenosine release from myocytes. The small effect on adenosine formation rates implies that the IMP-selective cytosolic 5'-nucleotidase plays a minor role in this tissue.
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PMID:Effect of 5'-deoxy-5'-isobutylthioadenosine on formation and release of adenosine from neonatal and adult rat ventricular myocytes. 848 9

To determine the capacity for purine nucleotide degradation among skeletal muscle fiber types, we established energy-depleted conditions in muscles of the rat hindlimb by inducing muscle contraction during ischemia. After 5, 10, 15, or 20 min of ischemic contractions, representative muscle sections were freeze-clamped and analyzed for purine nucleotides, nucleosides, and bases. Fast-twitch muscle sections accumulated about fourfold more IMP than the slow-twitch red soleus muscle. Inosine begins to accumulate at < 0.5 mumol/g IMP in slow-twitch muscle and at approximately 2 mumol/g IMP in fast-twitch muscle. This suggests that inosine is formed intracellularly by 5'-nucleotidase acting on IMP and that the activity and/or substrate affinity of the 5'-nucleotidase present in slow-twitch muscle may be higher than in fast-twitch muscle. At similar concentrations of precursor IMP, slow-twitch muscle has a greater capacity for purine nucleoside formation and should be more dependent on salvage and de novo synthesis of purine for the maintenance of muscle adenine nucleotides. Fast-twitch muscles are better able to retain IMP for subsequent reamination due to their lower capacity to degrade IMP to inosine.
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PMID:Purine nucleoside formation in rat skeletal muscle fiber types. 849 84

The major enzyme responsible for adenosine production during myocardial hypoxia or ischemia is 5'-nucleotidase. We purified an AMP-specific 5'-nucleotidase to homogeneity from the 150,000-g supernatant of dog heart homogenate using phosphocellulose, DEAE-cellulose, and ADP-agarose affinity chromatography. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified enzyme yielded a single protein band of 43 kDa. The molecular mass of the holoenzyme, determined by gel filtration and sucrose density-gradient centrifugation, was approximately 166 kDa, suggesting a tetrameric structure. Dog heart cytosolic 5'-nucleotidase was active at physiological pH (6.8-7.8) and demonstrated a preference for AMP over IMP as substrate. The enzyme exhibited sigmoidal saturation kinetics, with half-maximal activity at 2.6 mM AMP in the absence of ADP. ADP (0-250 microM) activated cytosolic 5'-nucleotidase by increasing maximal velocity and affinity for AMP. The enzyme was inhibited by 4 mM ATP, but 5'-nucleotidase activity increased as [ATP] was reduced. Mg2+ was required for activity, with maximal activation at approximately 3.5 mM free Mg2+. These data suggest that the regulation of AMP-specific cytosolic 5'-nucleotidase by adenine nucleotides and free Mg2+ may be important in the production of adenosine during conditions promoting ATP hydrolysis, such as myocardial hypoxia or ischemia.
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PMID:Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart. 849 66

Various 5'-nucleotidases (EC 3.1.3.5) exist in vertebrate tissues. The sequence and cDNA cloning of the membrane-bound ecto-5'-nucleotidase (e-N) and one of the cytosolic isoenzymes, IMP-preferring (c-N-II), but not the cytosolic AMP-preferring form (c-N-I), have been reported. While c-N-II has a broad tissue distribution, c-N-I is found only in vertebrate heart. The published data on substrate specificity involve mainly the naturally occurring nucleoside monophosphates, without a systematic structure-activity relationship study. In the present study we have used a series of AMP and IMP analogues to examine the structure-activity relationship for c-N-I and c-N-II in detail. The rank order of activity of the test compounds differed substantially between c-N-I and c-N-II. c-N-I and c-N-II varied with respect to the following interactions with substrate: (1) hydrogen-bond formation with the substituent in the 6-position of the purine ring (a donor-type with c-N-I and an acceptor-type with c-N-II); and (2) hydrophobic attraction of the 6-position unsubstituted purine ring (more pronounced with c-N-I than with c-N-II). No better substrate than 5'-AMP was found for c-N-I. We propose that c-N-I functions as an AMP-binding protein in the myocardial cell with an important role during ischaemic ATP breakdown when AMP accumulates rapidly.
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PMID:Structure-activity relationship of cytoplasmic 5'-nucleotidase substrate sites. 861 51

A soluble 5'-nucleotidase from pig thyroid was purified over 110-fold by chromatography on phosphocellulose, (NH4)2SO4 precipitation and gel filtration on Sephadex G-150. The purified 5-nucleotidase was free of non-specific phosphatases. The enzyme had optimum pH at 6.5 and hydrolysed preferentially IMP and GMP. The Km values were 0.66 and 1.0 mM for IMP and GMP, respectively. The enzyme also hydrolysed other nucleotides and showed the following relative Vmax:IMP>CMP>AMP>UMP.Mg2+ was necessary for the enzyme activity.
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PMID:Soluble 5'-nucleotidase from thyroid gland partial purification and properties. 861 79


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