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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)

Yeast plasmids have been constructed that carry the Herpes simplex Virus type 1 (HSV-1) thymidine kinase (TK) gene which is functionally expressed in Saccharomyces cerevisiae. The expression of the TK gene appears to be due to transcriptional read-through from a yeast promoter that lies on the 3' side of the HIS3 gene. The TK+ yeast possesses in vitro thymidine kinase activity which is absent in the original yeast strain. Yeast strains auxotrophic for thymidine monophosphate (dTMP) (tmp1) can grown on thymidine-containing medium after transformation with these plasmids. Tmp+, TK+ S. cerevisiae whose de novo synthesis of dTMP is inhibited with amethopterin plus sufanilamide is also capable of growth in thymidine. S. cerevisiae transformed with such plasmids is capable of incorporating thymidine and bromodeoxyuridine into DNA.
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PMID:Expression of the Herpes simplex virus thymidine kinase gene in Saccharomyces cerevisiae. 627 52

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

At 0 degrees C, CHO cells efficiently incorporated [3H]thymidine into the nucleotide fraction, but not into DNA. Upon reincubation of asynchronous cultures at 37 degrees C, 15-25% of the radioactivity contained in the cellular nucleotide fraction was released, in the form of thymidine, into the culture medium. At 0 degrees C, however, radioactivity of the nucleotide fraction was retained within the cells. Similarly, dTMP phosphatase (EC 3.1.3.35) in cell extracts was active at 37 degrees C, but not at 0 degrees C, whereas thymidine kinase (EC 2.7.1.21) was active at both temperatures. If synchronous cultures in Gl phase were prelabeled at 0 degrees C and reincubated at 37 degrees C, almost all radioactivity in the nucleotide fraction was released into the medium, whereas in S-phase cultures nearly all radioactivity of the nucleotide fraction was incorporated into DNA. In synchronous S-phase cultures treated with hydroxyurea, radioactivity in the nucleotide fraction was released into the medium at a rate considerably lower than that observed for Gl-phase cells. Rates of endogenous synthesis of thymidine nucleotides were calculated from changes of cellular thymidine nucleotide content, incorporation of thymidine nucleotides into DNA and release of thymidine into the medium during reincubation of prelabeled cultures in thymidine-free medium. The results obtained (see Table III) reveal marked differences between Gl and S phases with respect to the determinants of thymidine nucleotide metabolism.
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PMID:Thymidine nucleotide synthesis and catabolism by CHO cells and their changes during the cell cycle. 628 78

The deoxythymidine kinase (dTK) activity of a 5-methoxymethyldeoxyuridine-resistant mutant (MMdU(r)-20) of herpes simplex virus type 1 was compared with that of the parental wild-type (WT) virus. The dTK activity induced by the mutant was consistently less than that induced by the WT virus, was inhibited by antibody specific for herpes simplex virus dTK, and was more thermostable than the WT dTK. Further, it was inhibited to a lesser degree than the WT dTK by the nucleoside analogs MMdU and arabinosylthymine (araT), which suggests that one of the effects of the mutation was a selective alteration in substrate recognition by the dTK. The loss of ability to inhibit the mutant dTK by E-(2)-5-bromovinyldeoxyuridine was not as great as that seen with araT and MMdU. This agrees well with our previous observation that the MMdU(r)-20 mutant of herpes simplex virus is only partially resistant to this analog, as compared with araT and MMdU (V. Veerisetty and G. A. Gentry, Virology 114:576-579, 1981). [2-(14)C]araT was used to explore further the resistance to araT. Extracts of cells infected with the mutant, although producing a small amount of [(14)C]araTMP, were unable to produce [(14)C]araTTP, in contrast to extracts of cells infected with the WT virus. Both extracts, however, produced [(14)C]dTTP from [(14)C]deoxyribosylthymine. Finally, the ability of the extracts to phosphorylate [(14)C]dTMP was examined. It was found that this activity was greatly reduced relative to dTK activity in the case of the mutant. These findings suggest that a mutation in the dTK polypeptide has affected recognition not only of nucleoside substrates but of the nucleotide substrate dTMP as well, which agrees with the suggestion of Chen et al. that both activities are located on the same polypeptide (M. S. Chen and W. H. Prusoff, J. Biol. Chem. 253:1325-1327, 1978; M. S. Chen, J. Walker, and W. H. Prusoff, J. Biol. Chem. 254:10747-10753, 1979; M. S. Chen, W. P. Summers, J. Walker, W. C. Summers, and W. H. Prusoff, J. Virol. 30:942-945, 1979).
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PMID:Alterations in substrate specificity and physicochemical properties of deoxythymidine kinase of a drug-resistant herpes simplex virus type 1 mutant. 630 49

The herpes simplex virus(HSV)-coded thymidine kinase (TK) enzyme complex was isolated from HSV type 1 strain Lennette(TK+)-infected CLID (TK-) cells and was enriched by streptomycin sulfate and ammonium sulfate fractionation. This enzyme preparation was tested for dTMP:Ado (adenosine) and for dTMP:ADP phosphotransferase activities. The presence of dTMP:Ado phosphotransferase activity was proven by time-course studies with cells infected for 0-18 h, by biophysical studies in polyacrylamide gels, by affinity chromatography studies using AMP- and dTMP-Sepharose, and by immuno-neutralization experiments. The presence of dTMP:ADP phosphotransferase activity was demonstrated by kinetic experiments. These results were taken as evidence that the two functional subunits of the HSV-TK enzyme complex, AMP:dThd (deoxythymidine) phosphotransferase and ATP: dThd kinase, catalyze highly reversible enzyme reactions. New data are presented indicating that the ATP:dThd kinase is a nonspecific enzyme with respect to the nucleoside acceptor.
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PMID:Adenosine- and ADP-phosphorylating capacity of herpes simplex virus-induced thymidine kinase enzyme complex. 630 67

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

T4-infected cells, plasmolysed 15 min after infection, incorporate low concentrations (less than 20 microM) of deoxythymidine (TdR) into DNA at a significantly greater rate than dTMP, dTTP or thymine. At higher concentrations (greater than 40 microM), dTMP incorporation rate is high, approaching that of TdR at 200 microM. TdR is selectively incorporated at all concentrations tested, and is not inhibited by the other thymine containing DNA precursors. Incorporation of low concentrations of TdR requires the T4-induced thymidine kinase (tk) and is not significantly affected by the presence or absence of T4-induced thymidylate synthetase (td). We show that, in T4-infected plasmolysed cells, exogenously added TdR is preferentially incorporated into short DNA fragments during short pulse times. To explain these and other data a model is proposed in which thymidine plays a modulatory role between leading and lagging strand precursor feeds.
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PMID:Incorporation of thymine-containing DNA precursors in wild-type and mutant T4-infected plasmolysed cells. 635 61

The degree of inhibition of [3H]thymidine incorporation into DNA by exogenous deoxyuridine is assayed in a procedure known as the deoxyuridine suppression test. We report studies of the biochemical basis of this phenomenon in phytohemagglutinin-stimulated lymphocytes, which suggest that its mechanism has not been fully understood. Results show that inhibition by deoxyuridine is caused only in part by expansion of the intracellular pools of nonradioactive dTMP and dTTP, which dilutes the specific radioactivity of the [3H]dTMP and [3H]dTTP derived from [3H]thymidine. Increased dTTP levels also inhibit thymidine kinase. In addition, thymidine kinase is competitively inhibited by intracellular deoxyuridine. Inhibition of thymidine kinase activity by both mebolites further decreases the specific radioactivity of [3H]dTMP and [3H]dTTP. Deoxyuridine also inhibits the incorporation of [3H]deoxyadenosine and [3H]deoxyguanosine into DNA in these cells. Exogenous deoxyuridine still inhibits [3H]thymidine incorporation in cells whose de novo thymidylate synthesis has been strongly inhibited by 5-fluorodeoxyuridine or methotrexate. In such drug-treated cells, exposure to high concentrations of exogenous deoxyuridine can partially overcome the inhibition of thymidylate synthetase with resulting increase in the severely depleted dTTP pools. This increase is associated with enhanced DNA synthesis, as measured by incorporation into DNA of labeled deoxyribonucleosides other than [3H]thymidine. We conclude that exogenous deoxyuridine has multiple effects on [3H]thymidine incorporation, which must be considered in interpretations of deoxyurindine suppression test results.
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PMID:Biochemical mechanisms in the Killmann experiment: critique of the deoxyuridine suppression test. 644 7

Thymidine 5'-phosphate (TMP) derivatives with masked phosphate groups were synthesized in tritiated form from [methyl-3H]thymidine. They were of interest as models for 5' nucleotide derivatives that might be able to permeate mammalian cells and then liberate intracellular antimetabolite 5' nucleotides by loss of the masking groups. Mouse L fibroblasts were grown in vitro in the presence of 1 mM 5'-amino-5'-deoxythymidine, which was found to suppress greater than 99% of cellular thymidine kinase activity while inhibiting the rate of cell division by only 30%. The TMP derivatives were less effective than thymidine in labeling the deoxyribonucleic acid (DNA) of the L cells. The labeling was inhibited 95-99% by 5'-amino-5'-deoxythymidine, indicating that it represented incorporation into DNA of [3H]thymidine formed from degradation of the test compounds. No evidence was obtained that the compounds acted as sources of intracellular TMP by cell permeation followed by loss of phosphate blocking groups. Similar studies yielded no evidence that the bis(m-nitrophenyl) ester of TMP produced intracellular TMP by that route in the LM(TK-) strain of L cells that are genetically deficient in thymidine kinase.
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PMID:Bis(m-nitrophenyl) and bis(p-nitrophenyl) esters and the phosphorodiamidate of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in mouse cells in culture. 650 4

Cultured human alveolar macrophages (HAM phi) were found to produce soluble factors which inhibit tritiated thymidine [( 3H]-TdR) incorporation into tumour cells in vitro. We present evidence that thymidine (TdR) can be detected in HAM phi culture supernatants by thin-layer chromatography. Moreover, TdR secretion by HAM phi is an active process. Using [6-14C]-orotic acid as an early precursor to TdR and [3H]-TMP as the phosphorylation product, we were able to show that cultured HAM phi transformed them into labelled TdR. The very efficient thin-layer chromatography of the labelled metabolites was backed up by high-pressure liquid chromatography of nucleotides. HAM phi produce TdR by de novo synthesis and dephosphorylation. This phenomenon is due to the lack of thymidine kinase in normal mature macrophages. Since TdR, in high concentrations, can inhibit DNA synthesis through the 'TdR blockade' phenomenon, it is suggested that TdR secretion by HAM phi is a mechanism of non-specific modulation of tumour cell growth but highly restricted to the immediate macrophage microenvironment in vivo. The effectiveness of the thymidine gradient may thus be quite narrow, but is worthy of interest.
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PMID:Production of the effector molecule thymidine by human lung alveolar macrophages. 654 17


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