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)

Using gel filtration chromatography, we find a single peak of deoxythymidine phosphorylating activity in Chlamydomonas reinhardti. This activity has characteristics of a thymidine kinase, in that (1) it will utilize ATP (or dATP) or CTP (or dCTP) as phosphoryl donor, but not AMP or phenyl phosphate, and (2) it is inhibited by dTTP (and less so by dTDP, dUTP, and dUDP) but is unaffected by 3'-5' cyclic AMP. Partially purified chlamydomonas thymidine kinase has a pH optimum near 8.5, and a molecular weight of 80,000 to 85,000 daltons. Kinetic studies indicate a ping-pong mechanism with a Km for thymidine of 1.5 x 10(-7) moles per liter. 5-Bromo- and 5-fluorodeoxyuridine, and to a lesser degree deoxyuridine, are competitive inhibitors, but significant phosphorylation of these nucleotides could not be demonstrated in vitro by thymidine kinase. While thymidine is phosphorylated to dTMP by crude Chlamydomonas extracts, greater than 80% of the product formed by the partially purified enzyme is dTTP. Further, the gel filtration elution position of the single deoxythymidylate kinase activity present in cell extracts coincides with that of thymidine kinase. These results suggest that a multifunctional enzyme, rather than three separate phosphorylating activities, may be responsible for dTTP formation.
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PMID:Characterization of thymidine kinase and phosphorylation of deoxyribonucleosides in Chlamydomonas reinhardti. 4 38

The varicella zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) genes were cloned into the transcription vector pGEM4. In-vitro translation (ivt) of RNA transcribed from these genes showed prominent expression of functional TK proteins with the expected molecular weights of 36 kD for VZV and 43, 39, and 38 kD for HSV-1. The TK proteins were recognized by rabbit anti-VZV and anti-HSV-1 antibodies, respectively. Analysis of the ivt products by thin-layer chromatography revealed the conversion of thymidine to its phosphorylated forms (TMP, TDP, and TTP) by both the VZV and HSV-1 TK genes. The estimated specific activities of the in-vitro translated VZV and HSV-1 TKs were comparable. VZV TK templates were linearized at internal restriction sites and RNAs transcribed from these templates directed the synthesis of polypeptides with sizes consistent with the colinearity of the VZV TK gene. Deletion of 107 amino acids at the carboxy terminus of the VZV TK gene abolished the in-vitro TK activity. In addition, immunoprecipitation of truncated proteins synthesized in vitro suggested the possible involvement of the region between amino acid residues 101 and 168 from the amino terminus of the VZV TK molecule in the formation of structures necessary for antigenicity.
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PMID:In-vitro synthesis of functional varicella zoster and herpes simplex viral thymidine kinase. 169 24

Chronic exposure of H9 cells to 25 microM zidovudine (H9-AZT cells) causes a 2- to 3-fold increase in thymidine kinase (TK) activity (Agarwal RP, Int J Purines Pyrimidine Res, in press). The present study compared thymidine (TdR) and AZT anabolism in H9 and H9-AZT cells. After a 3.5-hr incubation with 10 microM TdR or AZT, the total intracellular accumulations of AZT (48.7 microM in H9 cells and 32.8 microM in H9-AZT cells) were 46.4% of TdR accumulation. Other major differences between TdR and AZT anabolism were: (i) the majority of TdR (84-87%) was incorporated into DNA compared to less than 1% of AZT; and (ii) whereas distribution of TdR in the nucleotides was TTP greater than TMP greater than TDP, zidovudine distributed was AZT-MP much greater than AZT-TP much greater than AZT-DP. Because of the poor substrate activity of AZT-MP for thymidylate kinase (TMP-kinase), most of the AZT (95-98%) remained as AZT-MP. TMP-kinase activities with TMP as substrate were 47.6 +/- 20.3 and 91.4 +/- 28.8 pmol/mg protein/min in H9 and H9-AZT cells, respectively. 5'-Nucleotidase activities with TMP as substrate were 428.9 +/- 37.8 and 255.9 +/- 28.7 pmol/mg protein/min in H9 and H9-AZT cells, respectively. Activities of these enzymes with AZT-MP as a substrate were very low. Despite an increase in TK and TMP-kinase, and a decrease in 5'-nucleotidase activities, the total intracellular accumulations of TdR and AZT were reduced significantly (P less than 0.05) to 67.5% in H9-AZT cells. Thymidine transport (0.66 to 0.68 pmol/sec/10(6) cells) was similar in both the cell lines. The severe reductions of TdR salvage caused by chronic exposure of cells to AZT, if it occurs in AIDS patients on AZT chemotherapy, may explain some of the long-term clinical toxicities of the drug.
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PMID:Thymidine and zidovudine metabolism in chronically zidovudine-exposed cells in vitro. 186 45

The naturally occurring nucleoside analogue arabinosyl thymidine is known as an anti-herpes and anti-cancer agent. The biologically active form is arabinosyl thymidine triphosphate (Ara-TTP), which inhibits cellular and viral DNA-polymerases and thus interferes with DNA replication. Using two murine erythroleukemia cell lines, Friend cell clone F4-6 and F4-12N, the latter being thymidine kinase deficient (TK-) cells transformed to a TK+ phenotype with the HSV TK gene, we have determined 1) the role of cellular and herpes simplex virus thymidine kinase (HSV TK) in the uptake of Ara-T into the cells; 2) the subsequent phosphorylation of intracellular Ara-T to Ara-TMP, Ara-TDP and Ara-TTP; 3) the incorporation of Ara-TTP into the DNA. Incorporation into DNA was studied under different conditions, including selective inhibition of the different cellular DNA polymerases by aphidicolin (that inhibits polymerases alpha and delta) and dideoxythymidine (that preferentially inhibits polymerases beta and gamma). The uptake of Ara-T into the methanol soluble pool of the cells depends upon its phosphorylation to Ara-TMP, which is more efficiently performed by the HSV TK than by the cellular TK, thus explaining the sensitivity of HSV infected cells to Ara-T. However, using increasing concentrations of Ara-T, we have shown that phosphorylation also occurs in normal control cells due to the cellular thymidine kinase. More than 90% of Ara-T is phosphorylated in the cell, and more than 60% of total Ara-T(MP, DP, TP) exists in the triphosphate form.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Arabinose furanosyl thymidine: uptake, phosphorylation and incorporation into DNA of mammalian cells. 196 22

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.
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PMID:Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast. 201 65

The effect of 3'-deoxythymidin-2'-ene (d4T) on the metabolism of exogenously supplied radiolabeled nucleosides was investigated. Following a 24-hr exposure to 250 microM d4T, we observed no significant effect on the incorporation of [3H]thymidine or [3H]deoxycytidine into DNA. In contrast, the amounts of [3H]uridine, [3H]deoxyuridine, and [3H]cytidine were significantly lower than those incorporated by control cultures. d4T had no significant effect on the incorporation of [3H]uridine or [3H]cytidine into RNA, or the incorporation of 3H-labeled amino acids into protein. In d4T-treated cells the relative proportions of [3H]dTMP, [3H]dTDP, and [3H]dTTP formed did not change but their absolute concentrations were increased. d4T significantly reduced the level of [3H]dUMP, and a parallel decrease in [3H]dTMP derived from [3H]dUMP was also evident. d4T increased the amounts of labeled deoxycytidine metabolites formed, with increased dCMP levels the most prominent. In a cell-free extract, [3H]d4T was phosphorylated at a rate of 1.6 pmol/30 min. Increasing concentrations of both thymidine and deoxyuridine inhibited the phosphorylation of [3H]d4T with IC50 values of 5.7 and 35 microM respectively. d4T was found to be a weak substrate for purified H9 cytosolic thymidine kinase (Km = 138 microM) and a weak competitive inhibitor of thymidine and deoxyuridine phosphorylation by this enzyme (Ki = 1.37 and 0.33 mM respectively).
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PMID:Effect of 3'-deoxythymidin-2'-ene (d4T) on nucleoside metabolism in H9 cells. 215 60

The effects of a variety of 5-, 5'-, and 3'-substituted deoxyuridine derivatives on the cytoplasmic thymidine kinase (EC 2.7.1.21) purified from a human colon carcinoma cell line, HCT 116, were determined. Of particular interest was elucidation of the structural features important for antagonism of the feedback inhibition of thymidine kinase exerted by thymidine triphosphate. Substitutions at the 5-position altered the potency of the 5'-modified compounds. The replacement of the 5-hydrogen with a methyl group or an iodine greatly increased the affinity of compounds for the thymidine kinase. This was evident for enzyme substrates with 5'-hydroxyl groups [2'-deoxyuridine (dUrd), 2'-deoxythymidine (dThd) and 5-iodo-2'-deoxyuridine (IdUrd)], feedback inhibitors with 5'-triphosphate substitutions (dUTP, dTTP and IdUTP), and for 5'-amino derivatives [5'-amino-2',5'-dideoxyuridine (5'-AdUrd), 5'-amino-2'-5'-dideoxythymidine (5'-AdThd) and 5-iodo-5'-amino-2',5'-dideoxyuridine (5'-AIdUrd)]. Qualitatively, however, the 5-substitutions did not affect the nature of the interactions with dThd kinase. For example, in the presence of dTTP, 5'-AdUrd stimulated dThd kinase activity as much as 5'-AdThd, but approximately a 100-fold greater concentration of 5'-AdUrd was required. Similar results were obtained using intact cells in which substitutions at the 5-position affected the potency, but not the efficacy, of the 5'-amino derivatives to stimulate dThd phosphorylation. In contrast, substitutions at the 5'-position did alter the nature of the interaction with dThd kinase. Thus, the 5'-hydroxyl compounds, dUrd, dThd and IdUrd, did not reverse the enzyme inhibition produced by dTTP nor did they stimulate dThd uptake in intact cells. 5'-Deoxy-5'-(ethylthio)thymidine, 5'-deoxy-5'-[(2-hydroxyethyl)thio]thymidine, and dTMP, but not dTDP, also antagonized the inhibition of dThd kinase produced by dTTP. In comparison to 5'-AdThd, the 3'-amino derivatives, 3'-AdThd and 3'-5'-diAdThd, were much less potent, but still efficacious, antagonists of feedback inhibition. These results indicate that a wide range of dUrd derivatives can disrupt the regulation of dThd kinase and provide leads for the development of new nucleotide analogues.
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PMID:Structure-activity analysis of antagonism of the feedback inhibition of thymidine kinase. 335 1

Vaccinia virus thymidine kinase activity is inhibited by low concentrations (10 microM) of dTDP or dTTP, but not by dTMP. This inhibition is specified for the thymidine nucleotides as dATP, dGTP, and dCTP have no effect. The viral enzyme phosphorylates thymidine to dTMP with typical first-order kinetics. However, evidence was obtained to indicate that the observed dTTP inhibition was noncompetitive in nature. This suggests that thymidine and dTTP interact with different sites in the native viral enzyme.
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PMID:Inhibition of vaccinia virus thymidine kinase by the distal products of its own metabolic pathway. 399 32

Cytoplasmic thymidine kinase from cardiac muscle of the rat has been characterized. It has a pH optimum of 9.0 and a K(m) value for thymidine of 1.6mum. The sedimentation coefficient of this enzyme in sucrose gradients is 4.5S, which represents a molecular weight of approx. 69000. Thymidine kinase prepared from cardiac muscle of foetal, neonatal and adult rats is inhibited by dTTP and dTDP; there is neither inhibition nor stimulation by dTMP, dCTP, dATP, dGTP or cyclic AMP. The activity of thymidine kinase in differentiating cardiac muscle of foetal and neonatal rats declines progressively with development, reaching adult values of almost zero by the fifteenth to seventeenth day of postnatal development. This represents a 70-fold decrease in enzyme activity from 3 days before birth to 17 days after birth. The loss of thymidine kinase activity in differentiating cardiac muscle correlates temporally with the cessation of DNA biosynthesis and the loss of cytoplasmic DNA polymerase activity in this tissue.
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PMID:Thymidine kinase activity in cardiac muscle during embryomic and postnatal development. 437 15

The activities of TdR kinase2 (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21), AdR kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76), GdR kinase (ATP: deoxyguanosine 5'-phosphotransferase, without EC number), ATP (Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3), nucleoside diphosphatase (nucleoside diphosphate phosphohydrolase, EC 3.6.1.6), nucleoside phosphotransferase (AMP: deoxynucleoside phosphotransferase, EC 2.7.1.77) and ribonucleotide 5'-diphosphate reductase (EC 1.17.4.1) were assayed in mitochondria of normal and regenerating rat liver. The activities of deoxynucleoside kinases are regulated: (a) by feedback inhibition of TdR kinase with dTTP and dCTP, and GdR kinase with dGTP; (b) GdR and AdR kinases by AdR and GdR inhibition, respectively; (c) by stimulation of GdR kinase with dTDP, dTTP and dATP. The stimulatory effects are correlated with changes of ATP (Mg2+)-ase and NDP-ase activities in regenerating liver mitochondria.
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PMID:Regulation of deoxynucleoside kinase activities in rat liver mitochondria. 612 3


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