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)

Methods have been developed to assay several aspects of 5-fluoro-2'-deoxyuridine and 5-fluorouracil metabolism in tissue culture cells. These methods allow measurement of (a) intracellular levels of the covalent complex formed between 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), 5,10-methylenetetrahydrofolate, and thymidylate synthetase; (b) incorporation of drug into RNA; and (c) analysis of drug metabolites. The methods were developed using radioactively labeled drugs, but they should be adaptable to studies using nonlabeled compounds. Sephadex G-25 chromatography or trichloroacetic acid precipitation were utilized for isolation of the macromolecular cell fraction; prior treatment of this fraction with RNase or heating at 65 degrees for 15 min resulted in selective removal of RNA or the thymidylate synthetase complex, respectively, from the precipitable fraction. Free intracellular drug metabolites present in the acid-soluble fraction were analyzed by high-pressure liquid chromatography. Following incubation of HTC cells with [6-3H]-5-fluoro-2'-deoxyuridine, a radioactive macromolecule was isolated and identified as a FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex. Intracellular formation of this complex was shown to be dependent on the presence of the enzyme thymidine kinase. Dissociation of the complex in vivo was first order with a t1/2 of 6.2 hr; in contrast, a t1/2 of 2 hr was determined for dissociation of the complex in cytosol. Incubation of L1210 cells with [6-3H]-5-fluorouracil for 22 hr resulted in formation of 80 fmol of FdUMP-5,10-methylenetetrahydrofolate-thymidylate synthetase complex per 10(6) cells, as compared with 400 fmol of drug incorporated into RNA per 10(6) cells. Intracellular FdUMP could not be detected in the acid-soluble fraction of these cells unless the cells were first heated to dissociate the complex.
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PMID:Assay of intracellular free and macromolecular-bound metabolites of 5-fluorodeoxyuridine and 5-fluorouracil. 15 5

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.
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PMID:Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria. 16 27

N1-S1/FdUrd Novikoff hepatoma cells, which lack thymidine kinase activity, are resistant to 5-fluorouracil (FUra) as well as 5-fluorodeoxyuridine (FdUrd), suggesting that the pathway, FUra leads to FdUrd leads to FdUMP, is utilized for the conversion of FUra to FdUMP. However, the inhibition of thymidylate synthetase activity, the presumed target of FdUMP, by 1 X 10(-4) M FUra in intact N1-S1 Novikoff hepatoma cells, which have significant levels of thymidine kinase activity, is completely eliminated by 5 X 10(-4) M hydroxyurea, which is a potent inhibitor of ribonucleotide reductase. These results imply that the formation of ribonucleotides and does not involve thymidine kinase. This apparent dichotomy can be explained by the fact that, in addition to the well known lack of thymidine kinase activity, [14C]FUra conversion to ribonucleotides is greatly depressed in the N1-S1/FdUrd cells. Hence, the formation of FdUMP from FUra in Novikoff hepatoma cells apparently proceeds primarily via the intermediate formation of ribonucleotides. The decreased conversion of FUra to ribonucleotides in N1-S1/FdUrd cells decreases not only the ability of the analog to inhibit DNA synthesis, but also its effect on RNA metabolism. FUra, at a concentration of 1 X 10(-5) M, inhibits rRNA maturation in N1-S1 cells, but not in N1-S1/FdUrd cells. Since N1-S1/FdUrd cells are completely resistant to 1 X 10(-5) M FUra, whereas N1-S1 cells are completely inhibited by 1 X 10(-5) M FUra, even in the presence of 1 X 10(-4) M thymidine, the effects of FUra on RNA metabolism appear to contribute significantly to the cytotoxicity of the analog at higher drug concentrations.
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PMID:Metabolism of 5-fluorouracil in sensitive and resistant Novikoff hepatoma cells. 19 Feb 17

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.
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PMID:Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions. 23 10

A method for the determination of relative values (%) of two pathways of thymidine-5'-phosphate (dTMP) formation, e.g. via de novo biosynthesis and through thymidine reutilization (salvage pathway), is proposed. It is shown that the relative values of dTMP formation through the salvage pathway in the mesometrial part of developing decidua in pregnant rats (9-11th day of ppregnancy) are 1.5-3.4 times higher as compared to those in the antimesometrial part. When dTMP biosynthesis is suppressed by aminopterine, up to 80% of total DNA thymind is synthesized at the expense of thymidine reutilization. The incorporation of 3H-thymidine into DNA was thereby increased approximately 8-fold irrespective of the decrease in the DNA synthesis rate (approximately 2.4 times). The dependence of the relative values of the thymidine reutilization pathway on the correlation of the thymidylate synthetase and thymidine kinase activities in the tissue is discussed. The ability of the cells to reutilize thymidine is interpreted in terms of their relative resistance to the effect of folic acid antagonists.
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PMID:[Determination of relative values of de novo biosynthesis and salvage pathway of thymidylate formation in rat decidual tissue]. 62 40

Three litters of 20 Wistar rat pups each were maintained until age 6 days at which time only the 4 lightest and 4 heaviest pups from each litter were left with the mother until age 13 days. Three control litters of eight pups each were also maintained for 13 days. At that time, the undernourished light pups showed body weight, cerebellar weight, and cerebellar DNA, respectively, of 79.2%, 86.6%, and 90.4% compared with a "combined control" group consisting of control pups plus undernourished heavy pups which were statistically indistinguishable with regard to these three measurements. After the week of "catch-up" or restorative body and brain growth, activities of enzymes from metabolic pathways leading to pyrimidine and nucleic acid biosynthesis were measured in cerebella from all three groups (control, undernourished heavy, and undernourished light). The salvage pathway enzyme thymidine kinase (TK) and the inter-conversion pathway enzyme thymidylate synthetase (TS) in the undernourished light group showed significant elevations of 32% and 11%, respectively, above activity in the combined control group. The salvage pathway enzyme uridine kinase (UK) and the de novo pathway enzyme aspartate transcarbamylase were not significantly different in cerebella from these two groups. The significant elevation in TK and TS in undernourished pups suggest that these enzymes are critical for restorative brain growth. The significant elevation of TS indicates that the inter-conversion pathway converting available uridylate, a ribonucleotide, to thymidylate, a deoxyribonucleotide, is activated in order to augment DNA biosynthesis.
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PMID:Pyrimidine metabolism during restorative brain growth after neonatal undernutrition in the rat. 84 83

Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.
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PMID:Deoxycytidine transport and pyrimidine deoxynucleotide metabolism in phytohaemagglutinin-stimulated pig lymphocytes. 93 56

The effects of 5-mercapto-2'-deoxyuridine (MUdr) on DNA synthesis in a primary murine spleen lymphocyte culture system stimulated by phytohemagglutinin (PHA) were studied. Inhibition of thymidine incorporation into acid insoluble nucleic acid material was 50% at 0.5 mM MUdR concentration, while inhibition of deoxyuridine incorporation into acid-insoluble nucleic acids was 50% at 0.01 mM MUdR. Time course studies, at 0.5 and 0.05 mM MUdR, showed that the magnitude of inhibition of incorporation for thymidine and deoxyuridine, respectively, increased from a time point after PHA stimulation when increased synthesis of thymidine kinase and thymidylate synthetase had leveled off. At 1 mM MUdR, total cellular DNA in cultures was decreased 43% at 42 hr after PHA stimulation. Neither the total number of cells nor the percentage of PHA-transformed cells was decreased in comparison to that of controls. MUdR therefore blocks the increase in DNA content of lymphocytes that is initiated during the S phase of the cell cycle. Millimolar levels of MUdR inhibited incorporation or uridine, adenosine, and cytidine into acid-insoluble material in pha-stimulated primary murine lymphocyte cultures. Total cellular RNA synthesis was inhibited at these levels of MUdR, with no differential effects on 4, 18, or 28 S RNA species observed. Uptake of these nucleosides into the total cellular acid-soluble material was not blocked. Uptake of different labeled nucleosides into cellular, acid-soluble pools occurs at different rates. Thus, choice of a suitable minimum pulse time to achieve saturation for different labeled nucleosides must relate to this consideration. Thymidine kinase from whole-cell sonic extracts of PHA-stimulated lymphocytes was inhibited 65% by 1 mM MUdR at 24 and 48 hr after stimulation. Uridine kinase extracted from the PHA-stimulated cells was also significantly inhibited by 1mM MUdR at 24 hr (56%). Exogenous guanosine incorporation into lympohcyte acid-insoluble material is increased by MUdR. This increased utilization of exogenous nuceloside is apparently the result of MUdR inhibition of conversion of adenosine to guanine nucleotides within the lymphocytes and a consequent diminution of the total intracellular guanine nucleotide pool size. The active inhibitory compound is the deoxyribonucleoside or deoxyribonucleotide. Comparison with the riboside analog 5-mercaptouridine showed that MUdR was a more efficient inhibitor of nucleoside incorporation.
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PMID:Effects of 5-mercapto-2'-deoxyuridine on the incorporation of nucleosides into RNA and DNA in a primary lymphocyte culture system. 97 90

DNA synthesis in the decidual tissue of rats on the 9-10th day of pregnancy has been studied in intact rats and under the effect of chloridin by means of radioautography and biochemical methods. The decidual cells of antimesometral and mesometral regions were shown to differ both by morphological features and intensity of 3H-thymidine utilization and activity of thymidylate synthetase and thymidine kinase. Under the conditions of the block of dihydropholate reductase, differences between the antimesometral and mesometral regions of deciduoma manifest themselves still more markedly. The decidual tissue consists of a heterogenous population of cells which differ by the ratio of different ways of thymidylate synthesis. An estimate is given for the ratio of two ways of thymidine monophosphate synthesis in the antimesometral regions of the decidual tissue.
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PMID:[DNA synthesis in the antimesometrial and mesometrial regions of rat decidual tissue]. 102 78

The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid). In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate. Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition. E. coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate. B. bacteriovourus grew at a normal rate on these depleted E. coli cells but with somewhat reduced cell yield. Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E. coli somewhat more than it inhibited growth on normal E. coli, but the effects were small compared with inhibition of axenic growth of the mutant. Total bdellovibrio DNA after growth on the depleted E. coli in the presence or absence of methotrexate exceeded the initial quanity of E. coli DNA present. Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA. The data are consistent with the prediction that intraperiplasmic growth of B. bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers. The data also indicate that B. bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth. The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B. bacteriovorus.
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PMID:Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus. 109 May 93


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