Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

5-AZA-2'-deoxycytidine-5'-monophosphate (5-AZA-dCMP) was tested as a substrate, and 5-aza-2'-deoxycytidine-5'-triphosphate (5-AZA-dCTP) was tested as an allosteric effector of purified spleen dCMP deaminase. Graphic analysis of the velocity of deamination of 5-AZA-dCMP versus its concentration gave a hyperbolic curve in which the estimated apparent Km was 0.1 mM. Since this curve was not sigmoidal and 5-AZA-dCMP at low concentrations stimulated the rate of deamination of the natural substrate, dCMP, it was proposed that the binding of 5-AZA-dCMP to the allosteric enzyme dCMP deaminase induced the R form. At substrate saturation, the rate of deamination of dCMP was 100-fold greater than that of 5-AZA-dCMP. dTTP inhibited the deamination of 5-AZA-dCMP with first-order kinetics. This inhibition was reversed by either 5-AZA-dCTP or dCTP. However, dCTP alone produced only a weak activation of the deamination of 5-AZA-dCMP in comparison to the potent activation when dCMP was the substrate. 5-AZA-dCTP was just as effective as dCTP for the allosteric activation of the deamination of dCMP. These results indicate that dCMP deaminase can play an important role in the metabolism 5-aza-2'-deoxycytidine nucleotides and may possibly modulate some of the pharmacological activity of this antimetabolite.
Mol Pharmacol 1984 May
PMID:Kinetic interaction of 5-AZA-2'-deoxycytidine-5'-monophosphate and its 5'-triphosphate with deoxycytidylate deaminase. 620 26

Intracellular pools of 5-fluoro-2'-deoxyuridine (FdUrd) and dUrd nucleotides were measured in cultured human lymphoblasts treated with FdUrd. At 1 microM FdUrd, intracellular 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was approximately 400 pmoles/10(6) cells, and FdUTP was approximately 0.1 pmole/10(6) cells. Intracellular dUMP and dUTP were elevated to values of approximately 1000 pmoles/10(6) cells and approximately 0.1 pmol/10(6) cells, respectively. With decrease in dTTP levels, utilization of FdUTP and dUTP as substrates for DNA synthesis became significant. FdUMP and dUMP, approximately 5 pmoles of each per micromole of DNA nucleotide, were found in DNA of cells treated with FdUrd (1 microM). The active removal of FUra and Ura from DNA of FdUrd-treated cells by the normal repair mechanism may lead to fragmentation of DNA and contribute to the cytotoxic effect of FdUrd.
Mol Pharmacol 1982 Jan
PMID:Nucleotide levels and incorporation of 5-fluorouracil and uracil into DNA of cells treated with 5-fluorodeoxyuridine. 621 71

5-Fluoro-2'-deoxyuridine at a concentration of 0.1 microM completely inhibited incorporation of radioactivity from [3H]dUrd into DNA in HeLa S3 cells within 5 min of exposure. The dTTP pool size decreased from 800 to 300 pmoles/10(7) cells by 1 hr, and then gradually increased after 3 hr of exposure. Although the incorporation of radioactivity from [32P] H3PO4 into DNA was inhibited, the relative incorporation into smaller fragments compared with larger fragments of DNA was found to be increased in the drug-treated cells. When the cells with [14C]thymidine-prelabeled DNA were exposed to 5 fluoro-dUrd, shifts of labeled DNA peaks to smaller sizes were observed between 5 and 18 hr after exposure, as analyzed by alkaline sucrose gradient centrifugation. Exposure of cells to 1 and 10 microM 5-fluoro-[3H]dUrd caused incorporation of radioactivity into DNA. By high-performance liquid chromatographic analysis of nucleosides, it was confirmed that almost all of the radioactivity was incorporated as 5-fluoro-dUrd.
Mol Pharmacol 1983 Jan
PMID:Effects of 5-fluoro-2'-deoxyuridine on DNA metabolism in HeLa cells. 622 4

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.
Mol Cell Biol 1983 Mar
PMID:Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells. 630 77

T4-infected cells, plasmolysed 15 min after infection, incorporate low concentrations (less than 20 microM) of deoxythymidine (TdR) into DNA at a significantly greater rate than dTMP, dTTP or thymine. At higher concentrations (greater than 40 microM), dTMP incorporation rate is high, approaching that of TdR at 200 microM. TdR is selectively incorporated at all concentrations tested, and is not inhibited by the other thymine containing DNA precursors. Incorporation of low concentrations of TdR requires the T4-induced thymidine kinase (tk) and is not significantly affected by the presence or absence of T4-induced thymidylate synthetase (td). We show that, in T4-infected plasmolysed cells, exogenously added TdR is preferentially incorporated into short DNA fragments during short pulse times. To explain these and other data a model is proposed in which thymidine plays a modulatory role between leading and lagging strand precursor feeds.
Mol Gen Genet 1983
PMID:Incorporation of thymine-containing DNA precursors in wild-type and mutant T4-infected plasmolysed cells. 635 61

Incorporation of TdR is aberrant in cells plasmolysed 15 min after infection by the recombination defective t4 chi and omega mutants. The in situ results parallel those obtained in vivo: at high TdR concentrations both T4 chi and T4 omega induced incorporation is slightly reduced compared to wild type, whereas at low TdR concentration incorporation induced by T4 chi is reduced and that induced by T4 omega is increased compared to wild type. No differences between wild type and mutant induced TdR incorporation are observed when cells are plasmolysed 8 min after infection. Further, no difference in incorporation between wild type and T4 chi or T4 omega is observed when either 3H thymine or 3H dTTP is used as a substrate, however small incorporation differences are observed using 3H dTMP as substrate. The mitomycin C sensitivity of T4 chi induced TdR incorporation is also observed in situ, but the drug must be present throughout infection. T4tk omega mutants have increased ability to incorporate 1 microM 3H TdR compared to T4tk and the reduced incorporation of 1 microM 3H TdR by T4 chi is suppressed in a T4td chi double mutant. These data are compatible with the hypothesis that endogenously produced TdR modulates leading and lagging strand synthesis and that the aberrant 1 microM TdR incorporation exhibited by T4 chi and T4 omega reflects specific activity changes resulting from a recombination defect induced alteration of the TdR "modulator pool".
Mol Gen Genet 1983
PMID:Incorporation of thymine-containing DNA precursors in plasmolysed cells infected by the T4 non-lethal recombination defective mutants. 635 62

The two thymidine kinases, TK 1 and TK 2, found in phytohemagglutinin-stimulated human lymphocytes and the thymidine kinase, TK 2N, found in unstimulated human lymphocytes were purified and characterized. All three kinases had molecular weights between 70 000 and 75 000 which increased to 170 000-200 000 in the presence of 2 mM ATP. Studies on the kinetic properties of the enzymes with thymidine and ATP as the substrates and dTTP as the inhibitor showed clear differences between TK 1 and TK 2, but a close similarity between TK 2 and TK 2N. With thymidine as the variable substrate, TK 1 showed Michaelis-Menten kinetics, whereas TK 2 and TK 2N showed characteristic biphasic kinetics. With ATP as the variable substrate, all three enzymes showed positive cooperative kinetics, but TK 2 and TK 2N lost the cooperativity in the presence of dTTP. The results from inhibition studies showed, that dTTP was a cooperative inhibitor of TK 1 but a non-cooperative inhibitor of TK 2 and TK 2N.
Mol Cell Biochem 1984 Sep
PMID:Differences in the kinetic properties of thymidine kinase isoenzymes in unstimulated and phytohemagglutinin-stimulated human lymphocytes. 650 22

A new protocol for inducing mutations in mammalian cells in culture by exposure to the thymidine analog 5-bromodeoxyuridine (BrdUrd) was established. This protocol, called "DNA-dependent" mutagenesis, involved the incorporation of BrdUrd into DNA under nonmutagenic conditions and the subsequent replication of the 5-bromouracil (BrUra)-containing DNA under mutagenic conditions but with no BrdUrd present in the culture medium. The mutagenic conditions were induced by allowing BrUra-containing DNA to replicate in the presence of high concentrations of thymidine. This generated high intracellular levels of dTTP and dGTP, causing nucleotide pool imbalance. The mutagenesis induced by this protocol was found to correlate with the level of BrUra substituted for thymine in DNA.
Mol Cell Biol 1984 Nov
PMID:Replication of DNA containing 5-bromouracil can be mutagenic in Syrian hamster cells. 651 25

Deoxycytidylate deaminase has been highly purified (1232-fold) from human leukemia CCRF-CEM cells. The native molecular weight of the enzyme is 108 000 and subunit molecular weight 50 500, suggesting that the native enzyme exists as a dimer. The enzyme exhibits a sigmoidal initial velocity vs substrate concentration curve and is regulated by allosteric effectors, dCTP and TTP. The curve relating substrate concentration to initial velocity was changed from a sigmoidal shape to a hyperbolic one by the activator dCTP, while the inhibitor TTP increased the sigmoidicity of the curve. The molecular weight of deoxycytidylate deaminase was unchanged in the presence of allosteric effectors, indicating that aggregation-disaggregation is not the basis of regulation. Deoxycytidylate deaminase exhibited the greatest affinity for the substrate dCMP, with lesser affinity for ara-CMP, and least affinity for CMP. Ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 4 microM. These data indicate that human neoplastic cell deoxycytidylate deaminase is a highly regulated allosteric enzyme, which is likely to have a significant influence on cellular dUMP, dCTP and TTP pools. These findings further suggest, that the enzyme through its influence on dUMP levels is likely to modulate the biochemical effects of pyrimidine antimetabolites active against the thymidylate synthetase reaction and in the presence of elevated dCTP pools will promote deamination of ara-CMP to the inactive ara-UMP.
Mol Cell Biochem 1983
PMID:Kinetic behaviour and allosteric regulation of human deoxycytidylate deaminase derived from leukemic cells. 658 81

3'-Amino-3'-deoxythymidine decreased the incorporation of [2-14C]thymidine into DNA of L1210 cells in vitro, and produced an accumulation of [2-14C]thymidine di- and triphosphate. The extent of these effects varied with the amount of recovery time after removal of 3'-amino-3'-deoxythymidine prior to addition of labeled thymidine. The distribution of radioactivity in the acid-soluble fraction derived from [3H]3'-amino-3'-deoxythymidine was as follows: 50% as 3'-amino-3'-deoxythymidine, 20% as the monophosphate, 10% as the diphosphate, and 20% as the triphosphate derivatives. No incorporation of [3H]3'-amino-3'-deoxythymidine into L1210 DNA could be detected. 3'-Amino-3'-deoxythymidine-5'-triphosphate is a competitive inhibitor against dTTP with a Ki of 3.3 microM, whereas the Km for dTTP was 8 microM using activated calf thymus DNA as the template and DNA polymerase-alpha. These data indicate that a major site of inhibition by 3'-amino-3'-deoxythymidine is inhibition of the DNA polymerase reaction.
Mol Pharmacol 1984 May
PMID:Molecular basis of the antineoplastic activity of 3'-amino-3'-deoxythymidine. 672 65


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