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)

5'-Nucleotidase, assayed as 5'-AMPase, has been extensively characterized and established as a stable, quantitative plasma membrane marker in HeLa S3 cells. The membrane 5'-AMPase has a Km of 7.0 microM. Relative affinities of the other 5'-mononucleotides for the enzyme are 5'-GMP > 5'-TMP > 5'-UMP > 5'-CMP. There are activity optima at pH7 and 10; the latter is Mg(2+)-dependent. The membrane preparations have a small amount of acid phosphatase activity that is distinct from 5'-AMPase activity but no alkaline phosphatase. AOPCP, ADP, and ATP are strongly inhibitory. Mg2+, Ca2+, or Co2+ additions do not affect the pH 7.0 activity; Mn2+ activates slightly, whereas Zn2+, Cu2+, and Ni2+ are inhibitory. EDTA slowly inactivates, but removal of the EDTA without the addition of divalent cations restores activity. The inactivation is also substantially reversed by Co2+ or Mn2+, but reactivability by divalent cations decreases with time in EDTA. ConA strongly inhibits, and alpha-methyl-D-mannoside or glucose (the latter much less efficiently) relieves the inhibition, indicating that the 5'-AMPase is a glycoprotein. Histidine is also inhibitory. Ouabain, phloretin, cytochalasin B, cysteine, phenyl-alanine, MalNEt, and IAA are without effect. 5'-AMPase activity codistributes with pulse-bound [3H]ouabain when either of two cell fractionation procedures are used. The 5'-AMPase activity per cell is constant at different cell densities in exponentially growing cells, and activity per unit cell volume remains constant throughout the cell cycle. These properties, together with its absence in other organelles, its stability to storage, its insensitivity to certain experimental manipulations, and its general insensitivity to inhibitors of specific transport systems, make 5'-AMPase a useful quantitative marker in studies on the regulation of HeLa membrane transport systems. Key Words: HeLa, 5'-nucleotidase, plasma membrane marker, non-specific phosphatases, divalent ions, ConA, AOPCP, cell cycle, mitochondria, transport inhibitors.
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PMID:Characterization of HeLa 5'-nucleotidase: a stable plasma membrane marker. 4 80

An increase of thymidine kinase [EC 2.7.1.21] activity and decrease of 5'-nucleotidase [EC 3.1.3.5] activity for dTMP were found during hormonal regeneration of the seminal vesicles by daily or single administration of testosterone propionate into mice castrated 2 weeks previously. Actinomycin D injected on day 0 of testosterone treatment completely inhibited both the increase of thymidine kinase and the decrease of 5'-nucleotidase. When injected on day 2, actinomycin D decreased thymidine kinase activity below the control level and 5'nucleotidase activity was not restored to the normal level. The activity of 5'-nucelotidase in a mixed sample, in which seminal vesicles of castrated mice and those of testosterone-treated mice were homogenized together, was intermediate between the activities determined separately. This indicates the absence of any inhibitor of 5'nucleotidase in the regenerating vesicles. Changes in total activity of 5'nucleotidase and total protein content in extracts during various treatments showed that the decrease in specific activity of 5'-nucleotidase in the first 2 days of testosterone treatment was not due to inhibition of enzyme activity but to dilution of the enzyme with other proteins which increased in content more rapidly than 5'-nucleotidase.
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PMID:Changes in enzyme activities of thymidine kinase and 5'-nucleotidase for dTMP during hormonal regeneration of seminal vesicles of mice. 7 66

Thymine-containing compounds, produced degradation of Escherichia coli DNA after infection of the cells with bacteriophage T5, did not accumulate in the cell but were excreted into the medium as the DNA was degraded. The ultimate degradation product was extracellular thymine that was not reutilized when T5 DNA synthesis began. This excretion of thymine may have been due in part to the induction of 5'-nucleotidase activity within 3 min after T5 infection. The level of this activity reached a maximum between 4 to 6 min after infection and then rapidly declined to its preinfection level by 10 to 15 min after infection. Chloramphenicol added before or soon after infection prevented the appearance of the nucleotidase. The induced nucleotidase activity was active not only on dTMP but also on dAMP, dGMP, and dCMP.
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PMID:Early events after infection of Escherichia coli by bacteriophage T5. Induction of a 5'-nucleotidase activity and excretion of free bases. 16 55

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.
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PMID:Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria. 22 41

A 5'-nucleotidase with unique specificity has been identified in the soluble fraction of normal human erythrocytes. It mediates the hydrolytic dephosphorylation of pyrimidine 5'-ribosemonophosphates but is catalytically ineffective with purine nucleotides or with the 2'-, 3'-, or cyclic isomers of pyrimidine nucleotides. Activities at 37 degrees in dialyzed hemolysates of nromal human erythrocytes averaged 7.3 and 6.2 mumol of Pi liberated per hour per g of hemoglobin for the substrates UMP and CMP, respectively. Activity with TMP as substrate was approximately one-half as much as with UMP or CMP. Apparent Michaelis constants were 0.33 mM UMP, 0.15 mM CMP, and 1.0 mM TMP. Magnesium was required for optimal activity, and this cation could not be replaced by Mn2+. Maximum activity was obtained between pH 7.0 and 7.5 with rapid decreases in more alkaline media and moderate decreases with acidification. The enzyme was quite sensitive to heat and was strongly inhibited by AMP, by some purine bases, and by both purine and pyrimidine nucleosides. Divalent cations of heavy metals were also strongly inhibitory, as were agents active against sulfhydryl groups. The presence of substrates and/or 2-mercaptoethanol provided considerable protection against some of these deleterious agents and conditions. Pyrimidine 5'-nucleotidase activity in hemolysates was clearly distinguishable from erythrocyte acid phosphatase and from leukocyte and serum alkaline phosphatases and nucleotidases.
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PMID:Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes. 24 Aug 46

Chronic exposure of H9 cells to 25 microM zidovudine (H9-AZT cells) causes a 2- to 3-fold increase in thymidine kinase (TK) activity (Agarwal RP, Int J Purines Pyrimidine Res, in press). The present study compared thymidine (TdR) and AZT anabolism in H9 and H9-AZT cells. After a 3.5-hr incubation with 10 microM TdR or AZT, the total intracellular accumulations of AZT (48.7 microM in H9 cells and 32.8 microM in H9-AZT cells) were 46.4% of TdR accumulation. Other major differences between TdR and AZT anabolism were: (i) the majority of TdR (84-87%) was incorporated into DNA compared to less than 1% of AZT; and (ii) whereas distribution of TdR in the nucleotides was TTP greater than TMP greater than TDP, zidovudine distributed was AZT-MP much greater than AZT-TP much greater than AZT-DP. Because of the poor substrate activity of AZT-MP for thymidylate kinase (TMP-kinase), most of the AZT (95-98%) remained as AZT-MP. TMP-kinase activities with TMP as substrate were 47.6 +/- 20.3 and 91.4 +/- 28.8 pmol/mg protein/min in H9 and H9-AZT cells, respectively. 5'-Nucleotidase activities with TMP as substrate were 428.9 +/- 37.8 and 255.9 +/- 28.7 pmol/mg protein/min in H9 and H9-AZT cells, respectively. Activities of these enzymes with AZT-MP as a substrate were very low. Despite an increase in TK and TMP-kinase, and a decrease in 5'-nucleotidase activities, the total intracellular accumulations of TdR and AZT were reduced significantly (P less than 0.05) to 67.5% in H9-AZT cells. Thymidine transport (0.66 to 0.68 pmol/sec/10(6) cells) was similar in both the cell lines. The severe reductions of TdR salvage caused by chronic exposure of cells to AZT, if it occurs in AIDS patients on AZT chemotherapy, may explain some of the long-term clinical toxicities of the drug.
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PMID:Thymidine and zidovudine metabolism in chronically zidovudine-exposed cells in vitro. 186 45

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

5'-Amino-2',5'-dideoxythymidine (5'-AdThd) is a nontoxic thymidine (dThd) analogue capable of antagonizing the feedback inhibition exerted by thymidine triphosphate (dTTP) on thymidine kinase (EC 2.7.1.21). In intact cells, this results in stimulation of thymidine uptake by 5'-AdThd. We have studied the interaction between 5'-AdThd and thymidine kinase purified from 647V cells. We found that 5'-AdThd inhibited competitively thymidine kinase activity (Ki of 0.5 microM) in the absence of dTTP whereas dTTP inhibited thymidine kinase activity in a noncompetitive manner. However, in the presence of dTTP, 5'-AdThd was able to stimulate enzyme activity in a mode that suggests competition with dTTP for the regulatory site. Altered interactions were observed at high substrate (dThd) concentrations, with dThd showing competitive kinetics with dTTP. In intact cells, we evaluated the hypothesis that antagonism of feedback inhibition could account for stimulation of dThd uptake by 5'-AdThd. If inhibition of thymidine kinase activity by dTTP is critical, then depletion of cellular dTTP by methotrexate should reduce the ability of 5'-AdThd to stimulate dThd uptake. Indeed, this was the case. If the dTTP pools were repleted by the addition of higher concentrations of dThd, the ability of 5'-AdThd to stimulate dThd uptake was restored. Furthermore, effects of 5'-AdThd on nucleoside phosphorylase or cytoplasmic 5'-nucleotidase activity (dTMP breakdown) could not account for the stimulation of dThd uptake in 647V cells. In summary, our results indicate that 5'-AdThd interacts with thymidine kinase at the dTTP-binding site, resulting in stimulation of enzyme activity and stimulation of dThd uptake in intact cells.
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PMID:Enzyme regulatory site-directed drugs: study of the interactions of 5'-amino-2', 5'-dideoxythymidine (5'-AdThd) and thymidine triphosphate with thymidine kinase and the relationship to the stimulation of thymidine uptake by 5'-AdThd in 647V cells. 253 72

We have previously reported that 5'-aminothymidine (5'-AdThd), an antagonist of the feedback inhibition exerted by dTTP that regulates thymidine kinase, enhances the uptake and cytotoxicity of 5-iododeoxyuridine in various human bladder cancer cell lines but not in normal human urothelial cells (HU) propagated in vitro. In this work we have analyzed the factors that could potentially account for the differential effect of 5'-AdThd among various cell types: 647V (a human bladder cancer cell line); HU; SV-HU (a SV40-transformed human urothelial cell line), and C3H/10T1/2 mouse embryo fibroblasts (10T1/2) cells. 5'-AdThd enhanced the uptake of IdUrd in SV-HU cells (greater than 400%), similar to what we have observed before for 647V cells. However, in 10T1/2 and HU cells, 5'-AdThd only minimally increased the uptake of 5-iododeoxyuridine (about 160%). Thymidine kinases purified from the different sources were similarly sensitive to inhibition by dTTP or 5'-AdThd and to deinhibition of the dTTP-induced regulation of enzyme activity by 5'-AdThd. Furthermore, [3H]-5'-AdThd permeated and accumulated intracellularly in all cell types. In none of these cultures was nucleoside phosphorylase activity detected, as indicated by the inability of the cells to produce thymine or iodouracil after exposure to the appropriate nucleosides. Also, 5'-AdThd did not affect the breakdown of dTMP by crude preparations of cytosolic 5'-nucleotidase from the different cells. We found that intracellular dTTP pools in the various cell types were substantially high (15-26 microM) compared to the sensitivity of thymidine kinase to inhibition by dTTP (IC50 2-4 microM). This suggests that thymidine kinase is in a strongly inhibited state in situ. To test the sensitivity of thymidine kinase (in situ) to regulation by dTTP we investigated: (a) the effect of depleting intracellular dTTP pools with methotrexate on the uptake of thymidine (dThd); and (b) the effect of pH on the uptake of dThd and its perturbation by 5'-AdThd, since the inhibition of thymidine kinase activity by dTTP is known to be pH dependent. We found that a 47% reduction of dTTP pools by methotrexate in 10T1/2 and HU cells did not result in an increase in thymidine kinase activity, as indicated by the lack of an effect on the uptake of dThd. However, we have previously shown that, under similar conditions, 647V cells show a substantial increase in dThd uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Basis for the differential modulation of the uptake of 5-iododeoxyuridine by 5'-aminothymidine among various cell types. 270 29

Residual 5'-nucleotidase activities in hemolysates from nine subjects with severe hereditary deficiency of pyrimidine nucleotidase (PyrNase) were compared to those in normal and reticulocyte-rich controls. Dephosphorylation rates of 12 potential ribo- and deoxyribomononucleotide substrates were measured as a function of pH. Data confirmed the existence of at least two isozymes of 5'-nucleotidase, PyrNase, and 2'-deoxy-5'-ribonucleotide phosphohydrolase (dNase) distinguishable by differences in maximal velocities, substrate preferences and restrictions, and pH optima. PyrNase was confirmed to be active principally with pyrimidine substrates (UMP = dCMP greater than CMP much greater than dTMP greater than dUMP) at a pH optimum of 7.5 +/- 0.1. dNase activity occurred with both purine and pyrimidine substrates and was maximal with deoxy analogs (dIMP much greater than dUMP greater than dGMP greater than dTMP = dAMP much greater than dCMP) at a pH optimum of 6.2, but slight cross-reactivity occurred with some nondeoxy substrates (IMP greater than GMP greater than UMP = XMP greater than CMP). PyrNase and dNase may be complementary systems that serve physiologically to clear the cytosol of RNA and DNA degradation products during maturation of erythroid elements by conversion of nucleotide monophosphates to diffusible nucleosides.
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PMID:Substrate specificity and pH sensitivity of deoxyribonucleotidase and pyrimidine nucleotidase activities in human hemolysates. 282 57


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