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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activites of the enzymes DNA polymerase, thymidine kinase, thymidylate kinase, thymidylate synthase, and deoxycytidylate deaminase have been measured in rat and human normal and neoplastic liver, in human fetal liver, and in cell lines derived from human hepatomas and rat transplantable hepatomas. The activities of these enzymes were increased in rat transplantable hepatomas, relative to rat normal or host liver, to a degree corresponding to the rapid growth rate of these tumors. With the exception of thymidine kinase, which did not change, the activities of these enzymes increased in human hepatomas relative to the corresponding host liver (apparently normal liver tissue from the same patient) and to human normal liver. The increases in enzyme activity observed in human hepatomas were small in comparison with those found in the rapidly growing rat hepatomas. The activities of deoxycytidylate deaminase in both human and rat liver tissues were 2 to 3 orders of magnitude higher than those of the other enzymes assayed. Activities of the enzymes of DNA synthesis in a slow-growing cell line derived from a human hepatoma were similar to those in human hepatoma tissues. In the case of rapidly growing cell lines derived from rat and human hepatomas, enzyme activities were higher than those in the corresponding tissues.
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PMID:Activities of some enzymes of pyrimidine and DNA synthesis in a rat transplantable hepatoma and human primary hepatomas, in cell lines derived from these tissues, and in human fetal liver. 624 89

Radioactive 5'-amino-5'-deoxythymidine (5'-AdThd), an inhibitor of herpes simplex virus, was synthesized by direct amination of monotosylated thymidine with concentrated ammonium hydroxide. Incubation of 5'-AdThd with purified herpes simplex virus type 1-encoded pyrimidine deoxyribonucleoside kinase produced the diphosphate derivative. The monphosphate derivative of 5'-AdThd was not detected as a reaction product of this enzymatic phosphorylation. A purified mixture of nonviral thymidine kinase and thymidylate kinase derived from uninfected Vero cells was unable to phosphorylate 5'-AdThd under identical conditions. The rate of hydrolysis of 5'-AdThd diphosphate increased as the pH of the reaction mixture decreased, with a shoulder region appearing between pH 3 and 5. The rate of hydrolysis was markedly increased below pH 3. 5'-AdThd, but not the monophosphate derivative, was detected in the hydrolysis mixture. The hydrolysis of 5'-AdThd diphosphate pH 3 also yielded some thymine in addition to 5'-AdThd.
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PMID:5'-Amino-5'-deoxythymidine: synthesis, specific phosphorylation by herpesvirus thymidine kinase, and stability to pH of the enzymically formed diphosphate derivative. 625 34

Recently we have described that the Herpes simplex virus (HSV)-induced thymidine kinase (TK) induces AMP- and ADP-dThd-5'-phosphotransferase activities. We now demonstrate the heterogeneity of the described activities in isoelectric focusing experiments and polyacrylamide gel electrophoresis. A TK--mutant of HSV type 1 fails to induce these activities. The activities of the type 1 enzyme complex was neutralized by an anti-HSV-serum. The TK-enzyme complex expressed in LTK--cells transformed to a TK+-phenotype by sheared HSV-1 DNA was compared with the wild type TK complex in isoelectric focusing experiments. Additionally we demonstrate that the HSV type 1 enzyme complex has thymidylate kinase activity, while the type 2 TK complex did not exhibit thymidylate kinase activity. Feedback regulation mechanisms by dTMP, dTDP and dTTP were investigated using partially purified enzyme preparations of HSV types 1 and 2 infected TK--cells.
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PMID:Analysis of the TK enzyme complex induced by HSV types 1 and 2 by means of isoelectric focusing and polyacrylamide gel electrophoresis. 628 60

The activities of the key enzymes of pyrimidine nucleotide and DNA syntheses in 43 human tumors and 28 normal human tissues were investigated. The activities of cytidine triphosphate synthetase, deoxycytidine monophosphate deaminase, uridine kinase, thymidine kinase, thymidine monophosphate kinase and DNA polymerase were markedly increased in tumor tissues, compared with those in the corresponding normal tissues, while the activities of deoxycytidine kinase, cytidine deaminase and deoxycytidine deaminase were only slightly increased. The use of thymidine and deoxyuridine as substrates of human pyrimidine nucleoside phosphorylase gave 1 to 2 orders of magnitude higher activity than that of uridine.
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PMID:Activities of various enzymes of pyrimidine nucleotide and DNA syntheses in normal and neoplastic human tissues. 628 2

Uptake and phosphorylation of exogenously supplied thymidine are stimulated in Strongylocentrotus purpuratus eggs after fertilization. Before fertilization, the rate of uptake is low and less than 10% of the thymidine entering the egg is phosphorylated. After fertilization, the rate of uptake increases over 50-fold and greater than 90% of the thymidine is immediately phosphorylated. These results imply that there is close cooperativity between fertilization-induced uptake and phosphorylation of thymidine. To gain insight into the structural basis of this apparent cooperativity and to provide a partial localization of the kinases, uptake and phosphorylation were measured in centrifuged eggs, and in centrifuged nucleate and anucleate merogons. Electron micrographs show that in these cells, the inner cytoplasmic contents are stratified according to density and displaced within the egg, whereas the outer cortical region of the cytoplasm remains intact. Uptake and phosphorylation of thymidine are fully stimulated in these eggs and merogons after fertilization, suggesting that both processes are mediated by an intact egg cortex. In support of this suggestion, we report that controlled disruption of the egg cortex prior to fertilization by treatment with cytochalasin B (CB) significantly reduces the rates of uptake and phosphorylation after fertilization. The full stimulation of phosphorylation in nucleate and anucleate merogons eliminates any localization of the catalyzing enzymes (thymidine kinase and thymidylate kinase) in the maternal nucleus and other inner cytoplasmic contents differentially segregated by centrifugation.
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PMID:Increased uptake of thymidine in the activation of sea urchin eggs. II. Cooperativity with phosphorylation, involvement of the cortex, and partial localization of the kinases. 630 15

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.
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PMID:Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line. 630 81

In HeLa and Vero cells the antiproliferative effects of iododeoxyuridine (IdUrd) were modulated in a biphasic manner by 5'-amino-5'-deoxythymidine (5'-AdThd). Low concentrations of 5'-AdThd increased the cytotoxicity of IdUrd whereas high concentrations of 5'-AdThd were antagonistic. Opposing effects on two enzymes, thymidine kinase (EC 2.7.1.21) and thymidylate kinase (EC 2.7.4.9), account for this unusual biphasic interaction. In the case of thymidine kinase, 5'-AdThd was found to antagonize the feedback inhibition which is normally exerted by the 5'-triphosphates of thymidine and IdUrd. Consequently, 5'-AdThd increased the rate of IdUrd phosphorylation. This stimulation (deinhibition) of enzyme activity was demonstrable in cell-free extracts and with a purified preparation of thymidine kinase provided that the 5'-triphosphates of IdUrd or thymidine were present. In their absence only enzyme inhibition was detected. In intact cells this stimulatory effect of 5'-AdThd was seen as a rapidly apparent, sustained increase in the steady-state levels of the phosphorylated IdUrd metabolites. As a result, IdUrd cytotoxicity was increased. Under these conditions, 5'-AdThd did not alter the relative abundance of the mono-, di-, and triphosphates of IdUrd. However, as the concentration of 5'-AdThd was raised, the percentage of IdUrd nucleotides present as iododeoxyuridylate increased dramatically. Corresponding reductions in the incorporation of IdUrd into cellular DNA and the associated cytotoxic effects were seen. These data suggested a second site of interaction, thymidylate kinase, the enzyme responsible for the conversion of iododeoxyuridylate to the diphosphate. In experiments measuring thymidylate kinase activity in cell-free extracts, 5'-AdThd effectively inhibited the phosphorylation of iododeoxyuridylate but not that of thymidylate. Additionally, 5'-AdThd did not produce an accumulation of thymidylate in intact cells. Thus, the ability of high concentrations of 5'-AdThd to antagonize the cytotoxicity produced by IdUrd without concomitantly inhibiting the phosphorylation of thymidylate and, thereby, reducing DNA synthesis was explained. Although the modulation of IdUrd metabolism produced by 5'-AdThd was qualitatively similar in Vero and HeLa cells, key quantitative differences were evident. Thus, 100 microM 5'-AdThd stimulated the uptake of 3 microM IdUrd in Vero cells but it was inhibitory in HeLa cells. Perturbation of nucleoside metabolism by agents such as 5'-AdThd may provide an important new way to achieve selective toxicity in cancer chemotherapy.
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PMID:Modulation of the metabolism and cytotoxicity of iododeoxyuridine by 5'-amino-5'-deoxythymidine. 686 13

3'-Azido-2',3'-dideoxy-5-iodouridine (AzIdUrd) and 3'-azido-2',3'-dideoxy-5-bromouridine (AzBdUrd), previously shown to be potent and selective inhibitors of human immunodeficiency virus replication in vitro were minimally toxic to the uninfected human lymphoid cell line H9 (IC50 = 197 and 590 microM, respectively). Both compounds strongly inhibited the incorporation of [3H]thymidine but not [3H]deoxyadenosine into DNA, and we observed no significant inhibition of [3H]uridine incorporation into RNA or [3H]amino acid incorporation into protein. Exposure of H9 cells to AzIdUrd or AzBdUrd (100 microM, 24 hr) and pulse-labeling with [3H]thymidine resulted in approximately 80% reduction in levels of tritiated dTMP, dTDP, and dTTP relative to control. [125I]AzIdUrd was phosphorylated rapidly in H9 cells with the monophosphate accounting for over 90% of total soluble radioactivity. A relatively low but stable level of AzIdUTP was maintained over a 12-hr period. [125I]AzIdUrd was phosphorylated by a cell free extract of H9 cells at a rate approximately three times that of thymidine and its phosphorylation was inhibited by excess thymidine. AzIdUrd was found to be a competitive inhibitor of cytosolic thymidine kinase with a Ki of 2.63 microM and AzIdUMP a weak competitive inhibitor of thymidylate kinase with a Ki of 55.3 microM. Both AzIdUTP and AzBdUTP were potent competitive inhibitors of HIV-1 reverse transcriptase (Ki = 0.028 and 0.043 microM, respectively) and relatively poor inhibitors of H9 cell DNA polymerase alpha (Ki = 42.0 and 42.7 microM, respectively). Thus, the high therapeutic index of these compounds is due to the sensitivity of the viral reverse transcriptase, coupled with the relative insensitivity of the host cell DNA polymerase alpha.
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PMID:Metabolism and mode of selective inhibition of human immunodeficiency virus replication by 3'-azido-2',3'-dideoxy-5-iodouridine and 3'-azido-2',3'-dideoxy-5-bromouridine. 767 40

We have reported that noncytotoxic concentrations of 3'-azido-3'-deoxythymidine (AZT) increase the cytotoxicity of ICI D1694, a folate-based thymidylate synthase (TS) inhibitor, with increasing AZT incorporation into DNA. We postulated that the inhibition of TS by ICI D1694 would decrease 5'-deoxythymidine triphosphate (dTTP) pools, which compete with AZT triphosphate (AZT-TP) as a substrate for DNA polymerase. Furthermore, the inhibition of TS would increase the activity of both thymidine kinase (TK) and thymidylate kinase (TdK). Each of these consequences of TS inhibition would favor more incorporation of AZT into DNA. Thus, we reasoned that other TS inhibitors should also result in increased AZT incorporation into DNA and, perhaps, in increased cytotoxicity. N6-[4-(Morpholinosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz[ cd]indole glucuronate (AG-331) differs from ICI D1694 in that it is a de novo designed lipophilic TS inhibitor, it does not require a specific carrier for cellular uptake, and it does not undergo intracellular polyglutamation. This potent TS inhibitor causes minimal cytotoxicity in MGH-U1 human bladder cancer cells. A 24-h exposure to 5 microM AG-331 causes almost complete TS inhibition but only 35% cell kill. The combination of AZT and AG-331 in MGH-U1 cells resulted in an enhanced antitumor effect relative to that of each agent alone; 50 microM AZT, noncytotoxic alone, increased the cell kill of induced by AG-331 from 35% to 50%. Biochemical studies of this combination revealed that simultaneous treatment with 5 microM AG-331 plus 1.8 microM [3H]-AZT produced as much as a 68% +/- 7% increase in AZT incorporation into DNA. This observation was associated with an increase in DNA single-strand breaks, measured as comet tail moment, of up to 6.6-fold. These studies support our original premise that TS inhibition favors increased incorporation of AZT into DNA and that the combination causes more cell kill than either drug alone in MGH-U1 cells.
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PMID:Cytotoxic and biochemical implications of combining AZT and AG-331. 785 Sep 19

The mechanism responsible for the decreased sensitivity of a clinical herpes simplex virus type 1 (HSV-1) isolate, HSV-145, to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was examined. Measurements of 50% inhibitory doses of several drugs demonstrated that although HSV-145 was sensitive to phosphonoacetic acid, adenine arabinoside and acyclovir, its sensitivity to BVDU and 5-(2-chloroethyl)-2'-deoxyuridine was significantly less than that normally observed for HSV-1. Analysis of the thymidylate kinase (TMP-K) activity of HSV-145 thymidine kinase (TK) demonstrated a decreased level of TMP-K activity when compared to HSV-1 TK. The TMP-K activity of HSV-145 resembled that observed for HSV-2 and the TK-deficient strain HSV-1 TK-7. When the nucleotide sequence of the HSV-145 TK gene was compared to that of the HSV-1 strains C1(101) and SC16 a single nucleotide substitution (G changed to A at base position 502) was detected which would result in the substitution of threonine at amino acid position 168 for alanine. The substitution is the same as that for the laboratory-derived BVDU-resistant virus HSV-1 SC16B3. Collectively, these studies highlight the importance of amino acid conservation at position 168 of the HSV-1 TK in conferring efficient TMP-K activity and BVDU sensitivity.
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PMID:Analysis of the thymidine kinase of a herpes simplex virus type 1 isolate that exhibits resistance to (E)-5-(2-bromovinyl)-2'-deoxyuridine. 802 3


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