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)

Four cell lines biochemically transformed by u.v.-irradiated herpes simplex virus contain virus DNA fragments ranging from 3 to 22% of the HSV genome. Of five revertant clones selected for 3H-TdR or BrdUrd resistance, four had lost all detectable virus DNA while the fifth, selected for BrdUrd resistance, retained the entire virus fragment but there was a reduction of virus copies per cell from 5 to 1. Three 'supertransformed' revertant cell lines contained virus DNA fragments ranging from 12 to 28%. The number of virus DNA fragments per cell ranged from 1 to 5 and clearly indicated that a single copy of the virus thymidine kinase gene is adequate for biochemical transformation. The determination of the base composition of the transforming virus DNA fragment indicated that the transforming DNA has a base composition approximately the same as the HSV genome and does not constitute a low GC virus DNA region. Cross hybridization between HSV-1 transformed cells and HSV-2 DNA is very slight, indicating that the DNA found in clone 139 is not entirely composed of the HSV-1 and HSV-2 common sequences.
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PMID:Quantification of the herpes simplex virus DNA present in biochemically transformed mouse cells and their revertants. 19 37

We have studied the expression of mouse galactokinase in human-mouse somatic cell hybrids segregating mouse chromosomes. Since concordant segregation of the expression of mouse galactokinase and the presence of mouse chromosome 11 were observed in the hybrid clones, we conclude that the gene for mouse galactokinase is located on mouse chromosome 11. We have also investigated the expression of mouse galactokinase in somatic cell hybrids between thymidine kinase-deficient Chinese hamster cells and mouse peritoneal macrophages. The results of this study indicate that the expression of mouse galactokinase and thymidine kinase segregates concordantly, and, therefore, we infer that the gene for mouse thymidine kinase is also located on mouse chromosome 11.
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PMID:Synteny of the genes for thymidine kinase and galactokinase in the mouse and their assignment to mouse chromosome 11. 19 14

The incubation of a cell-free protein-synthesizing system prepared from rabbit reticulocytes with cytoplasmic RNA from herpes simplex virus (HSV)-infected cells resulted in increased thymidine kinase activity. This enzyme activity was specifically inhibited by anti-HSV antiserum and was relatively unaffected by TTP, an inhibitor of cellular thymidine kinases. Induction of the new activity was prevented by addition of inhibitors of eucaryotic protein synthesis, and no new activity was detected when RNA from cells infected with pyrimidine deoxyribonucleoside kinase-deficient mutants, instead of wild-type HSV, was added. An increased deoxycytidine kinase activity with similar properties to the HSV-specified enzyme activity was also present in cell-free systems incubated with RNA from HSV-infected cells. Phosphorylation of thymidine and deoxycytidine at 30 degrees C continued for longer than 11 h. The findings are consistent with the accurate synthesis in vitro of enzymically active HSV-specified pyrimidine deoxyribonucleoside kinase.
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PMID:Cell-free synthesis of herpes simplex virus-coded pyrimidine deoxyribonucleoside kinase enzyme. 19 56

1-beta-d-Arabinofuranosylthymine (ara-T), a metabolite of the sponge Tethya crypta, has shown selective activity against herpes simplex virus (HSV) replication (G. A. Gentry and J. F. Aswell, Virology 65:294-296, 1975). Analysis of HSV-infected and uninfected cell lysates by CsCl isopycnic centrifugation showed that ara-T blocked the incorporation of [(3)H]hypoxanthine into viral deoxyribonucleic acid and, to a large extent, into host deoxyribonucleic acid of infected (but not uninfected) cells. Additional experiments with [gamma-(32)P]adenosine 5'-triphosphate as a radiophosphate donor demonstrated that ara-T is phosphorylated by extracts of HSV-infected BHK cells and not by those of uninfected cells. At an ara-T concentration that almost completely inhibited the growth of LM cells, which had been transformed to a pyrimidine deoxyribonucleoside kinase(+) (dPyK(+)) phenotype by ultraviolet-inactivated HSV-1, the growth of uninfected LM cells was not affected. These results indicate that the viral dPyK is responsible for the selective antiviral activity of ara-T. This conclusion was further supported by experiments that showed that the replication of a variety of dPyK(-) mutants of HSV-1 and HSV-2 were not affected by ara-T and that ara-T inhibited the phosphorylation of deoxycytidine and deoxythymidine by HSV-1 dPyK, but not by host deoxycytidine and deoxythymidine kinases, respectively. Ara-T also selectively inhibited the replication of equine herpesvirus type 1 (EHV-1) in vitro and was effective against EHV-1 infection in vivo in hamsters. Further, EHV-1 was inhibited by ara-T and by bromodeoxyuridine in LM cells lacking a cytosol thymidine kinase, suggesting that EHV-1 induces a dPyK. Finally, spectrophotometric assay for thymine suggested that ara-T is not a substrate for nucleoside phosphorylase of hamster liver, and a microbiological assay indicated that substantial amounts of ara-T were excreted in the urine of uninfected hamsters that had received a single injection of 5 mg of ara-T, the amount given in each injection in the in vivo experiments with EHV-1.
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PMID:Antiviral activity of arabinosylthymine in herpesviral replication: mechanism of action in vivo and in vitro. 19 86

[125I]deoxycytidine was a good substrate for herpes simplex virus type 1 thymidine kinase (TK), whereas [125I]deoxycytidine was a very poor substrate for cellular TK. Simple, sensitive, and specific assays for viral TK could be carried out in vivo and in vitro even in the presence of cell TK. Autoradiographic detection of incorporated [125I]deoxycytidine provided a rapid and simple method for detection of and screening for viral TK in infected as well as viral TK-transformed cells.
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PMID:[125I]deoxycytidine used in a rapid, sensitive, and specific assay for herpes simplex virus type 1 thymidine kinase. 19 81

To better understand the pathogenesis of infantile viral gastroenteritis, we studied Na+ and Cl- fluxes in vitro in short-circuited jejunal epithelium from 8-10-day-old piglets after infection with a standard dose of human rotavirus given via nasogastric tube. 11 infected piglets, all of whom became ill, were compared with 9 uninfected, healthy litter-mates. When killed 72 h after infection, intestinal villi were shorter and crypts deeper (P less than 0.025) in duodenum, upper jejunum, and mid-small intestine, but not ileum in infected piglets. Virus antigen was seen by fluorescence microscopy in occasional jejunal villus tip cells in only four infected piglets and no controls at 72 h. Net Na+ and Cl- fluxes did not differ from noninfected litter-mate controls under basal conditions, but response to glucose was blunted in infected piglets (P less than 0.001). Theophylline stimulated net Cl- secretion in both infected and control animals, and cyclic AMP concentration in isolated jejunal villus enterocytes did not differ significantly. In isolated jejunal villus enterocytes of infected piglets, thymidine kinase activity increased (P less than 0.001), and sucrase activity decreased (P less than 0.001). We conclude that in this invasive enteritis caused by a major human viral pathogen, glucose-coupled Na+ transport is impaired in the jejunum at a time when the villus epithelium shows enzyme characteristics of crypt epithelium, and when little or no virus is present. These findings are identical to those occurring in an invasive coronavirus enteritis of piglets but differ markedly from those seen with enterotoxigenic diarrhea.
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PMID:Human rotavirus enteritis induced in conventional piglets. Intestinal structure and transport. 19 22

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.
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PMID:Detection of herpes simplex virus thymidine kinase polypeptides in cells labeled with 35S-methionine. 20 86

Incorporation of thymidine into Novikoff rat hepatoma cells was analyzed with a rapid sampling technique which allowed collection of 12 time points in 20 sec. Transport was a rapid, saturable, nonconcentrative process with a Km of about 85 micrometer. The intracellular thymidine pool was also rapidly labeled in cells which phosphorylated thymidine, that a group translocation process involving thymidine kinase can be ruled out. Under all conditions examined, phosphorylation, not the transport, of thymidine was the rate-determining step in its incorporation into the acid-soluble pool. Estimation of transport rates from total incorporation into cells which phosphorylate the substrate is invalid in this cell system and must be questioned in all instances.
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PMID:Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling technique. 20 8

A guanine derivative with an acyclic side chain, 2-hydroxyethoxymethyl, at position 9 has potent antiviral activity [dose for 50% inhibition (ED(50)) = 0.1 muM] against herpes simplex virus type 1. This acyclic nucleoside analog, termed acycloguanosine, is converted to a monophosphate by a virus-specified pyrimidine deoxynucleoside (thymidine) kinase and is subsequently converted to acycloguanosine di- and triphosphates. In the uninfected host cell (Vero) these phosphorylations of acycloguanosine occur to a very limited extent. Acycloguanosine triphosphate inhibits herpes simplex virus DNA polymerase (DNA nucleotidyltransferase) 10-30 times more effectively than cellular (HeLa S3) DNA polymerase. These factors contribute to the drug's selectivity; inhibition of growth of the host cell requires a 3000-fold greater concentration of drug than does the inhibition of viral multiplication. There is, moreover, the strong possibility of chain termination of the viral DNA by incorporation of acycloguanosine. The identity of the kinase that phosphorylates acycloguanosine was determined after separation of the cellular and virus-specified thymidine kinase activities by affinity chromatography, by reversal studies with thymidine, and by the lack of monophosphate formation in a temperature-sensitive, thymidine kinase-deficient mutant of the KOS strain of herpes simplex virus type 1 (tsA1).
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PMID:Selectivity of action of an antiherpetic agent, 9-(2-hydroxyethoxymethyl) guanine. 20 61

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.
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PMID:Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus. 20 89


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