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 have undertaken to characterize the role of cytoplasmic 5'-nucleotidase (EC 3.1.3.5) in the phosphorylation of the anti-herpes simplex virus and anti-human cytomegalovirus agent ganciclovir (GCV) in MOLT-4 cells, a human T cell line adapted to grow in suspension culture. The rate of formation of GCV triphosphate was found to be approximately doubled by preincubation of nontransfected MOLT-4 cells with agents that cause the accumulation of IMP, such as ribavirin (20 microM) and mycophenolic acid (1 microM), and the reaction rate was found to be unaffected by high levels of thymidine (100 microM). With herpes simplex virus-1 thymidine kinase (HStk) gene-transduced MOLT-4 cells, the rate of GCV phosphorylation was approximately 40-fold faster than that in uninfected cells and, in marked contrast to uninfected cells, the reaction was significantly inhibited both by IMP dehydrogenase inhibitors and by thymidine. These latter effects appear to be the result of 1) the accumulation of high levels of dTTP in IMP dehydrogenase inhibitor-treated cells, with consequent feedback inhibition of HStk, and 2) direct competitive substrate inhibition by thymidine of the HStk-catalyzed phosphorylation of GCV. Thus, agents that enhance 5'-nucleotidase-catalyzed phosphorylation of GCV in uninfected cells do not play a similar role in HStk-transfected cells, a consequence of the quantitative predominance of the viral thymidine kinase-catalyzed reaction over that attributable to endogenous cytoplasmic 5'-nucleotidase.
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PMID:Effects of IMP dehydrogenase inhibitors on the phosphorylation of ganciclovir in MOLT-4 cells before and after herpes simplex virus thymidine kinase gene transduction. 791 Mar 73

The anti-human immunodeficiency virus agents 2',3'-dideoxyadenosine (ddAdo) and 2'-beta-fluoro-2',3'-dideoxyadenosine (2'-beta-F-ddAdo) are rapidly converted, both in vitro and in vivo, to the corresponding inosine analogs by the widely distributed enzyme adenosine deaminase (EC 3.5.4.4). We have determined the effects of the potent adenosine deaminase inhibitor 2'-deoxycoformycin (2'-dCF) on ddAdo and 2'-beta-F-ddAdo metabolism in MOLT-4 cells and on ddAdo antiviral activity in the ATH8 test system. At levels as low as 5 nM in the incubation medium, 2'-dCF effectively blocks the extracellular deamination of both agents, thus permitting their rapid cellular uptake as the unchanged parent compounds, rather than as the less lipid-soluble 2',3'-dideoxyinosine or 2'-beta-fluoro-2',3'-dideoxyinosine. The result is a significant increase in intracellular levels of the pharmacologically active forms 2',3'-dideoxyadenosine-5'-triphosphate and 2'-beta-fluoro-2',3'-dideoxyadenosine-5'-triphosphate. The effect becomes maximal over the range of 50-250 nM 2'-dCF and declines to control levels when extracellular 2'-dCF levels exceed 1 microM. This decrease in ddAdo and 2'-beta-F-ddAdo phosphorylation with higher levels of the inhibitor appears to result from intracellular penetration of 2'-dCF and consequent inhibition of intracellular deamination, a critical step in the activation of both agents through the 5'-nucleotidase pathway. In anti-human immunodeficiency virus assays, a 2.2-fold increase in ddAdo antiviral potency was seen at 2'-dCF levels of 20 and 50 nM.
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PMID:Enhancement by 2'-deoxycoformycin of the 5'-phosphorylation and anti-human immunodeficiency virus activity of 2',3'-dideoxyadenosine and 2'-beta-fluoro-2',3'-dideoxyadenosine. 796 62

The specific activity of phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) and inositol 1,4,5-trisphosphate monophosphatase (IP3-MP) involved in phosphoinositide catabolism was found to be significantly lower in the total homogenate of four human lymphoblastoid cell lines, HSB-2, MOLT-4, CEM and JURKAT, than in resting and activated peripheral blood mononuclear cells, ranging from 0.8 to 3.2 and from 1.3 to 3.7 nmol/min/mg for PIP2-PLC and IP3-MP, respectively. In PHA-stimulated cells, the specific activities were enhanced 25 and 35% respectively over the values (8.02 and 7.83 nmol/min/mg, respectively) measured in resting peripheral blood mononuclear cells. After centrifugation on discontinuous sucrose gradient of cell homogenate, PIP2-PLC and IP3-MP activities were found to be predominantly associated with the cytosol fraction (> 69%) in HSB-2 and MOLT-4 cells, with a distribution similar to that found in PHA-stimulated and in resting lymphocytes. In CEM cells, about half of the total activity remained in this fraction, while in JURKAT lymphoblastic cells more than 45% of the total activity was recovered in the high-density membrane fraction (d = 1.20-1.25), the soluble PIP2-PLC and IP3-MP activity accounting for only 13 and 25%, respectively. Conversely, in less differentiated leukemic cells HSB-2 and MOLT-4, conspicuous activity of the ectoenzymes 5'-nucleotidase (5'-NT) and gamma-glutamyltransferase (gamma-GT) was recovered in the soluble fraction. Growing leukemic cells at a distinct level of differentiation have a general reduction in activity but a characteristic distribution of enzymes involved in the transmission of signals usually targeting the cell surface.
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PMID:Subcellular localization of inositide enzymes in established T-cell lines and activated lymphocytes. 809 39

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 carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.
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PMID:Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase. 959 24

The relative levels of the deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), and the 5'-nucleotidase (5'-NT) are of importance for the effect of many nucleoside analogues used in the treatment of hematological malignancies. To elucidate dCK, dGK and 5'-NT gene expressions in cell lines and in samples from patients with leukemia, we have established a real-time quantitative PCR (RQ-PCR) method. From the available dCK, dGK and 5'-NT cDNA sequences we designed specific primers and fluorogenic probes for the respective genes. The mRNA of dCK, dGK and 5'-NT was also measured by semi-quantitative RT-PCR, the enzyme activities by a radioactive substrate-based technique and Western blot was used to measure the amount of dCK and dGK protein. A MOLT-4 wild-type and its 9-beta-D-arabinofuranosylguanine (Ara-G)-resistant subline was used for the methods comparisons and the RQ-PCR assay was used in 35 samples from pediatric patients with ALL and AML. The results from RQ-PCR for the cell lines were in agreement with the semi-quantitative RT-PCR. The mRNA expression for dCK, dGK and 5'-NT (expressed as the ratio of the respective gene and the reference gene) in pediatric ALL and AML patients showed a large interindividual variability from 0.06 to 2.34, non-detectable to 0.06 and 0.04 to 0.30, respectively. These results show that the quantitative evaluation by RQ-PCR is a valuable tool in the determination of dCK, dGK and 5'-NT mRNA levels in cell lines and in clinical samples which were expressed at various levels. This rapid, convenient and specific method is suitable for further studies of these genes in clinical samples.
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PMID:Real-time quantitative PCR assays for deoxycytidine kinase, deoxyguanosine kinase and 5'-nucleotidase mRNA measurement in cell lines and in patients with leukemia. 1189 43