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)

Dialyzed extracts of Bacillus megaterium KM contain thymidine, deoxyadenosine, and deoxyguanosine kinase activities. Thymidine kinase activity is best with deoxyadenosine triphosphate or deoxyguanosine triphosphate (dGTP) as the phosphoryl donor, whereas the best deoxyadenosine kinase activity is obtained with dGTP or adenosine triphosphate. Deoxyguanosine kinase activity functions optimally with deoxycytidine triphosphate as the donor. Although the thymidine kinase activity of crude extracts does not have a demonstrable divalent cation requirement, the addition of Mg(2+) or Mn(2+) is necessary for the formation of thymidine di- and triphosphates. The synthesis of thymidine kinase appears to be partially derepressed by thymine starvation. Incubation of extracts with deoxyadenosine and dGTP results in the substantial accumulation of deoxyadenosine di- and triphosphates. Extracts deaminate deoxycytidine to deoxyuridine, presumably as a consequence of the action of deoxycytidine deaminase, and then convert deoxyuridine to deoxyuridylic acid. B. megaterium extracts do not contain any detectable deoxycytidine kinase activity.
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PMID:Deoxynucleoside kinases of Bacillus megaterium KM. 499 37

The biochemical basis of cellular resistance to 9-beta-D-arabinofuranosyladenine (ara-A) and its natural purine derivative, deoxyadenosine, was investigated with two mutants of cultured human T-lymphoblastoid CCRF-CEM cells. One mutant that lacked deoxycytidine kinase activity, designated CEM/ara-C, retained about 10% of wild-type deoxyadenosine kinase and deoxyguanosine kinase activity each but maintained normal adenosine kinase or thymidine kinase activity. This suggested that in these human T-lymphoblastoid cells, as in other previously studied mammalian cells, deoxycytidine and purine deoxyribonucleosides are phosphorylated by the same enzyme. Despite this extensive reduction of purine nucleoside kinase activities, the cytotoxicity of ara-A or deoxyadenosine was not appreciably affected, decreasing by only 2.5- and 6-fold, respectively. A second mutant, isolated by selecting CEM/ara-C mutants that were resistant to ara-A, showed a 100-fold increase in resistance to ara-A cytotoxicity. This ara-A-resistant subline was deficient in the activities of two enzymes, deoxycytidine kinase and adenosine kinase, and showed a high degree of resistance to deoxyadenosine, adenosine, and pyrazofurin but not to pyrimidine analogs, such as 5-fluorodeoxyuridine or 5-fluorouridine. Further studies of ara-A and deoxyadenosine phosphorylation in wild-type and resistant cell lines disclosed that, although deoxycytidine kinase is the principal enzyme for their phosphorylation in vitro, their intracellular conversion to cytotoxic nucleotides depends on the joint action of deoxycytidine kinase and adenosine kinase rather than purine-specific deoxynucleoside kinase, as previously thought.
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PMID:Identification of the mechanism of activation of 9-beta-D-arabinofuranosyladenine in human lymphoid cells using mutants deficient in nucleoside kinases. 627 78

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

The specific activity of thymidine kinase (TK) was higher in spleen than in thymus or unseparated tonsillar lymphocytes, while deoxycytidine kinase (dCK) specific activity was lowest in spleen and was much higher in thymus and in unseparated tonsillar lymphocytes. The ratio of dCK to TK was always high in thymus, in unseparated and in B-cell-enriched tonsillar lymphocytes (between 2 and 5), but it was always low in spleen (0.3-0.4). The difference in the pyrimidine nucleoside phosphorylating enzyme activities of the thymus and spleen does not seem to be a mere consequence of different DNA synthesis rates, because the activities of DNA polymerase-alpha were practically the same in these organs. Unseparated and B-cell-enriched tonsillar lymphocytes resemble the thymus with respect to the ratio of dCK to TK activities, while the T-cell-enriched fraction contained 3-5 times lower activities of both enzymes. These results suggest that the metabolic pathways of CdR and TdR utilization for DNA synthesis differ in the lymphocyte populations independently from their rate of DNA polymerization and they may be in connection with their maturation processes.
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PMID:Differences between lymphoid organs with respect to the phosphorylation of deoxycytidine and thymidine. 630 28

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] (ACV), a potent antiviral compound, was phosphorylated to the same extent by extracts from untreated and iododeoxyuridine-treated Epstein-Barr virus-containing latent D98/HR-1 somatic hybrid cells. ATP was the preferred phosphate donor over other nucleoside triphosphates. The cytosol extract from D98/HR-1 cells effected optimum phosphorylation of thymidine at pH 8.0, whereas ACV was phosphorylated equally well over a wide pH range. Electrophoretic analysis of thymidine kinase-, deoxycytidine kinase-, and ACV-phosphorylating activities from both untreated and iododeoxyuridine-treated cell extracts displayed identical properties. A small part (5 to 10%) of the loaded ACV-phosphorylating activity seemed to migrate with the deoxycytidine kinase activity from cytosol. dTTP and dCTP, at relatively high concentrations, partially inhibited ACV-phosphorylating activity. The results suggest that Epstein-Barr virus does not code for its own thymidine kinase and that phosphorylation of ACV in Epstein-Barr virus-producing cells is carried out by multiple or as yet unidentified ATP-dependent nonspecific cellular phosphotransferases.
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PMID:Phosphorylation of acyclovir in vitro in activated Burkitt somatic cell hybrids. 631 70

5-Fluoro-2'-deoxycytidine (FdCyd) is a potent inhibitor of growth of tissue culture cells. The major cytotoxic event appears to be inhibition of thymidylate synthetase as evidenced by reversal of the cytotoxicity with thymidine but not deoxycytidine and by the effect of FdCyd on nucleotide pools, which is characteristic of specific inhibition of this enzyme. The metabolism of FdCyd was established by using a method in which its cytotoxicity was compared in several S-49 mutant cell lines having defined single or double deficiencies of enzymes involved in nucleoside and nucleotide metabolism. Our results indicate that FdCyd is metabolized to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of thymidylate synthetase by two pathways: (i) sequential reactions catalyzed by deoxycytidine kinase and deoxycytidylate deaminase and (ii) sequential reactions catalyzed by cytidine deaminase and thymidine kinase. We have shown that metabolism of FdCyd can be directed through the former pathway by inhibition of cytidine deaminase with tetrahydrouridine. Since cytidine deaminase appears to be responsible for catabolism of FdCyd in animals, our results suggest that the antineoplastic effects of FdCyd should be examined in combination with inhibitors of cytidine deaminase.
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PMID:Metabolism and mechanism of action of 5-fluorodeoxycytidine. 695 27

The current study was undertaken to determine the relevance of leukemic blast cell proliferative activity, cellular parameters of Ara-C metabolism and the in vitro sensitivity to GM-CSF in association with the clinical response to TAD-9 induction therapy in 66 patients with de novo acute myeloid leukemia (AML). Proliferative activity was assessed by 3H-thymidine (3H-TdR) incorporation and thymidine kinase (TK) activity, parameters of Ara-C metabolism comprised the activities of deoxycytidine kinase (DCK) and DNA polymerase alpha (poly alpha) as well as Ara-CTP concentrations and 3H-Ara-C uptake into DNA. GM-CSF sensitivity was determined by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4 h concurrent exposure to GM-CSF and 3H-TdR (0.5 microCi/ml). The following results were obtained as expressed by median values and ranges: 3H-TdR incorporation: 1.07 pmol/10(5) cells (0.0-10.1), TK: 7.3 pmol/min/mg protein (1.3-56.0), DCK: 9.3 pmol/min/mg protein (0.77-47.1), poly alpha: 1.7 pmol/min/mg protein (0.00-28.9), Ara-CTP: 53.3 ng/10(7) cells (13.3-211.0), 3H-Ara-C uptake: 0.06 pmol/10(5) cells (0.0-0.57). 3H-Ara-C uptake was correlated with 3H-TdR incorporation (r = 0.74) and with the (S-phase dependent) activities of TK (r = 0.73) and poly alpha (r = 0.71, but not with DCK activity or intracellular Ara-CTP content. Blast cells of 37 from 55 analyzed patients were found to be sensitive to GM-CSF stimulation as defined by an increase in 3H-TdR incorporation > or = 1.5-fold over control values after the 48 h GM-CSF exposure. In vitro data were related with clinical response to TAD-9 induction therapy in 43 patients with newly diagnosed AML, taking the blast cell reduction at day 10 or 16 to < 5% or > or = 5% residual blasts as early parameter for adequate or inadequate response, respectively. While neither 3H-Ara-C uptake, nor intracellular Ara-CTP concentration, TK nor DCK activity were predictive for response, a high 3H-TdR incorporation and a high poly alpha activity were associated with adequate blast cell reduction. Median values of 3H-TdR incorporation were 2.26 pmol/10(5) cells for patients with adequate blast cell clearance and 0.80 pmol/10(5) cells for patients with inadequate blast cell clearance (P = 0.11), the respective values for poly alpha were 3.22 pmol/min/mg protein for responders and 1.1 pmol/min/mg protein for non-responders (P = 0.0085).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Blast cell proliferative activity and sensitivity to GM-CSF in vitro are associated with early response to TAD-9 induction therapy in acute myeloid leukemia. 747 75

The mammalian deoxyribonucleoside kinases are deoxycytidine kinase, thymidine kinase 1 and 2 and deoxyguanosine kinase. These enzymes phosphorylate deoxyribonucleosides and thereby provide an alternative to de novo synthesis of DNA precursors. Their activities are essential for the activation of several chemotherapeutically important nucleoside analogues. In recent years, these enzymes have been thoroughly characterised with regard to structure, substrate specificity and patterns of expression. In this review, these results are reviewed and furthermore, the physiologic metabolic role of the anabolic enzymes is discussed in relation to catabolic pathways. The significance of this information for the development of therapeutic protocols and choice of animal model systems is discussed. Finally, alternative pathways for nucleoside analogue phosphorylation are surveyed, such as the phosphotransfer capacity of 5'-nucleotidase.
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PMID:Mammalian deoxyribonucleoside kinases. 749 63

Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.
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PMID:Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26. 789 98

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.
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PMID:De novo synthesis of pyrimidine nucleotides by Helicobacter pylori. 792 75


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