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)

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.
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PMID:Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes. 115 5

Male mice of 7 different strains were injected i.p. with 400 mg/kg of butylated hydroxytoluene (BHT). 2 and 4 days later, the incorporation of thymidine into pulmonary DNA was significantly increased in all treated animals and this was accompanied by an increase in lung weight and pulmonary DNA. Thymidine kinase activity and DNA polymerase activity were enhanced in the lungs of BHT-treated animals and maximum activity of these enzymes appeared to precede maximum thymidine incorporation by 24 h. 3 days after BHT a good correlation was found between administered dose and thymidine kinase activity. Measuring the activity of this enzyme might serve as a convenient biochemical marker to follow and to quantitate BHT-produced cell proliferation in lung. The concentrations of cyclic AMP and the activity of adenylate cyclase were not altered by BHT on days 1-9 after administration. BHT produced also some dose-dependent, time-dependent increases in the activities of pulmonary 5'-nucleotidase and glucose-6-phosphate dehydrogenase (G6PDH), but had little effect on isocitric dehydrogenase (ICDH), pyruvate kinase (PK) and lactic dehydrogenase (LDH).
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PMID:Biochemical paramters of BHT-induced cell growth in mouse lung. 124 55

Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man.
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PMID:Differentiation associated changes in thymidylate synthetase and thymidine kinase activities in intestinal cells. 126 Aug 61

Roswell Park Memorial Institute 4265 human lymphoblasts were grown with three dihydrofolate reductase inhibitors: a 2,4-diaminopteridine, methotrexate; a 2,4-diaminoquinazoline, chlorasquin; and, a 2,4-diaminotriazine, triazinate. In the absence of inhibitor, dihydrofolate reductase activity increased to a peak at mid-log growth and then declined during the later growth stages. When cells were grown with 10(-8) M antifolate, cell growth was not affected, but dihydrofolate reductase activity (assayed at pH 7.0) remained at approximately initial levels throughout the growth cycle. This represented 60 to 70% less activity at the mid-log stage of growth, as compared to control cells. Dihydrofolate reductase activity in cells grown with 10(-8) M methotrexate, when assayed at pH 8.5, reached levels twice those in control cells. Enzyme activity in cells grown with 10(-8) M chlorasquin, when assayed at pH 8.5, was also higher than at pH 7.0, but it was not as high as that observed in methotrexate-treated cells. Activity in cells grown with 10(-8) M triazinate was approximately the same when assayed at either pH 7.0 or 8.5. At 10(-8) M, the three antifolates had no effect on the activities of thymidylate synthetase, thymidine kinase, serine trans-hydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, and thymidylate kinase. However, when concentrations were used which completely inhibited growth (10(-7) to 10(-5) M methotrexate or chlorasuin; 10(-6) to 10(-5) M triazinate), dihydrofolate reductase was progressively inhibited, and there was a two- and a threefold elevation of thymidylate synthetase and thymidine kinase activity, respectively. Quantitatively, the elevation of either enzyme was similar over the range of growth-inhibitory concentrations studied. The activities of the other enzymes were unaffected. Methotrexate and chlorasquin inhibited thymidylate synthetase in a noncompetitive manner (with respect to 5,10-methylenetetrahydrofolate) with approximate Ki values of 4.5 X 10(-5) M and 4.9 X 10(-6) M, respectively. Triazinate, at 10(-3) M, had no significant effect on thymidylate synthetase activity. At 10(-3) M, the antifolates produced a negligible inhibition of thymidine kinase. Deoxyuridine 5'-monophosphate (10(-5) M) effectively protected thymidylate synthetase from heat inactivation in vitro. Dihydrofolate or 5,10-methylenetetrahydrofolate, at 10(-3) M, only partially protected thymidylate synthetase. Concentrations of methotrexate (10(-7) to 10(-6) M), chlorasquin (10(-7) M), and triazinate (10(-6) to 10(-5) M), which produced thymidylate synthetase elevation in vivo, did not protect the enzyme from heat inactivation in vitro. Methotrexate at 10(-5) M and chlorasquin at 10(-6) M gave slight protection. Thymidine kinase was stabilized only by thymidine.
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PMID:Elevation of dihydrofolate reductase, thymidylate synthetase, and thymidine kinase in cultured mammalian cells after exposure to folate antagonists. 127 51

Human cytomegalovirus (HCMV, a betaherpes virus) is the cause of serious disease in immunologically compromised individuals, including those with acquired immunodeficiency syndrome. One of the compounds used in the chemotherapy of HCMV infections is the nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (ganciclovir). The mechanism of action of this drug is dependent on the formation of the nucleoside triphosphate, which is a strong inhibitor of the viral DNA polymerase. Thymidine kinase, which is encoded by many of the herpesviruses, catalyses the initial phosphorylation of ganciclovir. But there is no evidence for the coding of this enzyme by HCMV, and DNA sequence analysis of the HCMV genome has shown that there is no open reading frame characteristic of a herpesvirus thymidine kinase. Here we present biochemical and immunological evidence that the HCMV UL97 open reading frame codes for a protein capable of phosphorylating ganciclovir. This protein seems to be responsible for the selectivity of ganciclovir and will be useful tool in the understanding and refinement of the antiviral activity of new selective anti-HCMV compounds.
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PMID:Human cytomegalovirus UL97 open reading frame encodes a protein that phosphorylates the antiviral nucleoside analogue ganciclovir. 131 59

Constant exposure of mastocytoma P-815 cells to adenosine 3',5'-cyclic decylphosphoramidate (1), which is permeable to the cell membrane and resistant to the action of phosphodiesterase, caused a dose-dependent (1 to 50 microM) inhibition in the synthesis of DNA and cell proliferation. Pretreating the cells with compound 1 (20 microM, 4 h) caused considerable inhibition of the incorporation of [3H]thymidine ([3H]TdR) into [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) and that of [14C]hypoxanthine into nucleic acid, but not the synthesis of [14C]dTTP from [U-14C]aspartate. These results indicate that compound 1 preferentially inhibits the salvage synthesis of intracellular nucleotides and nucleic acids. Thymidine kinase, a key enzyme in salvage synthesis of nucleotides, was almost undetectable in cells pretreated with compound 1 at 20 microM for 4 h or at 5 microM for 15 h. On the other hand, compound 1 activated partially purified cAMP-dependent protein kinase A from bovine heart. Judging from these observations, it is likely that compound 1 readily permeates the cell membrane, activates cAMP-dependent protein kinase, then inhibits the salvage synthesis of nucleotides and nucleic acids by inhibiting thymidine kinase, which results in the inhibition of cell growth.
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PMID:Inhibition of salvage synthesis of nucleic acid by adenosine 3',5'-cyclic decylphosphoramidate in mastocytoma P-815 cells. 133 57

The activities of thymidine kinase and uridine kinase (enzymes for pyrimidine salvage pathway) in phytohemagglutinin (PHA)--prestimulated lymphocytes were inhibited by arginase in a similar pattern to the inhibition on thymidine incorporation. Further study revealed that arginase did not directly affect the activities of these enzymes in the cell-free system. Thymidine kinase and uridine kinase activities of PHA-prestimulated lymphocytes were inhibited by arginase making their activities as low as that cultured in arginine-free RPMI-1640 medium. These results suggest that arginine-depletion in the culture medium is the primary mode of action of arginase on the inhibition of mitogen-stimulated lymphocyte proliferation.
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PMID:Inhibition of lymphocyte proliferation by liver arginase. 143 81

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.
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PMID:Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain. 159 87

Aedes albopictus mosquito cells resistant to the thymidine analog 5-bromodeoxyuridine (BrdU) were obtained using a single-step selection procedure. The resistant cells were characterized with respect to growth in the presence of BrdU, incorporation of [3H]uridine and [3H]thymidine, and thymidine kinase activity in crude extracts. The LC50 for TK-6 cells was 95 micrograms BrdU/ml, and for TK-8 cells was 45 micrograms/ml. Both clones incorporated [3H]uridine at levels corresponding to those in wild-type cells. TK-6 and TK-8 cells did not incorporate [3H]thymidine into acid-precipitable material, nor did they contain measurable thymidine kinase activity. Thymidine kinase activity in crude extracts from wild-type cells had a Km of 2 microM and a Vmax of 10 pmol.min-1.mg-1 protein.
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PMID:Thymidine kinase-deficient mutants of Aedes albopictus mosquito cells. 163 44

Thymidine kinase (TK), which is induced by Herpes Simplex Virus 1 (HSV1), plays a key role in the antiviral activity of guanine derivatives such as aciclovir (ACV). In contrast, ACV shows only low affinity to the corresponding host cell enzyme. In order to define the differences in substrate binding of the two enzymes on molecular level, models for the three-dimensional (3-D) structures of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites of HSV1-TK and human TK were developed. The reconstruction of the active sites started from primary and secondary structure analysis of various kinases. The results were validated to homologous enzymes with known 3-D structures. The models predict that both enzymes consist of a central core beta-sheet structure, connected by loops and alpha-helices very similar to the overall structure of other nucleotide binding enzymes. The phosphate binding site is made up of a highly conserved glycine-rich loop at the N-terminus of the proteins and a conserved region at the C-terminus. The thymidine recognition site was found about 100 amino acids downstream from the phosphate binding loop. The differing substrate specificity of human and HSV1-TK can be explained by amino-acid substitutions in the homologous regions. To achieve a better understanding of the structure of the active site and how the thymidine kinase proteins interact with their substrates, the corresponding complexes of thymidine and dihydroxypropoxyguanine (DHPG) with HSV1 and human TK were built. For the docking of the guanine derivative, the X-ray structure of Elongation Factor Tu (EF-Tu), co-crystallized with guanosine diphosphate, was taken as reference. Fitting of thymidine into the active sites was done with respect to similar interactions found in thymidylate kinase. To complement the analysis of the 3-D structures of the two kinases and the substrate enzyme interactions, site-directed mutagenesis of the thymidine recognition site of HSV1-TK has been undertaken, changing Asp162 in the thymidine recognition site into Asn. First investigations reveal that the enzymatic activity of the mutant protein is destroyed.
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PMID:Computer-aided active-site-directed modeling of the herpes simplex virus 1 and human thymidine kinase. 166 55


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