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)

We have studied the effect of pH on the interactions between thymidine kinase, thymidine triphosphate, and 5'-amino-2',5'-dideoxythymidine (5'-AdThd) in purified preparations of the enzyme and in intact 647V cells, a human bladder cancer cell line. Thymidine kinase is competitively inhibited by 5'-AdThd. dTTP feedback inhibits in a noncompetitive fashion. However, 5'-AdThd partially reverses the inhibition produced by dTTP resulting in enhanced enzyme activity. We have found that dTTP (pKa = 7.5) is a much more potent inhibitor of purified preparations of thymidine kinase activity at low pH conditions. For example, 2.5 microM dTTP inhibited thymidine kinase activity by 50, 85, and 95% at pH values of 8.0, 7.5, and 6.5, respectively. The interaction of 5'-AdThd (pKa = 8.5) at either the active (competitive) or the regulatory (deinhibition) site is not altered significantly over a pH range of 6.5 to 9.5. To extend these findings to intact cells, we studied the perturbation of the uptake of thymidine by 5'-AdThd in 647V cells incubated in media buffered at various pH values. In cells exposed to media buffered at pH 8.5 or 7.5, 5'-AdThd maximally stimulated thymidine uptake about 250 and 300% at 10 and 30 microM, respectively. However, at pH 6.5, 300 microM 5'AdThd was required to produce maximal stimulation of about 500%. These observations are consistent with the greater sensitivity of thymidine kinase (in situ) to feedback inhibition by dTTP at the lower pH conditions. Intracellular dTTP pool sizes were not affected by variations in pH during the short time course of our experiments. However, after 1 h, the intracellular concentration of 5'-AdThd was twice that of the extracellular medium in conditions at pH 7.5 and 8.5 but was equimolar across the membrane at pH 6.5. This does not account for the differences in the perturbation of thymidine uptake by 5'-AdThd at various pH values. In general, our results indicate that regulation of thymidine kinase by dTTP is pH dependent, while its modulation by 5'-AdThd is not, and that regulation of thymidine kinase in situ is sensitive to alterations in pH.
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PMID:Modulation of the feedback regulation of thymidine kinase activity by pH in 647V cells. 279 Jul 82

The experiments described in the present work were designed to study the function of the N-terminal end of thymidine kinase (TK) encoded by herpes simplex virus type 1. Specifically we were interested to know whether this end was involved in binding of the enzyme to other molecules, had any influence on its subcellular localization or affected one or more of the activities associated with the enzyme. A parental enzyme and a deletion mutant, lacking the 45 N-terminal amino acids, derived from this strain, were used. Thymidine kinase from the parental virus bound to DNA-Sepharose, but the truncated enzyme did not. This was apparently not due to a specific ability to bind to DNA, since immunofluorescence studies indicated that both the normal and the deleted TK were mainly located in the cytoplasm, preferentially in the perinuclear region. Phosphorylation of thymidine as well as the amounts of TK polypeptides were markedly reduced at late times after infection with the mutant, but not to the same extent after infection with the wild-type. The deleted TK gene was efficiently transcribed as shown by hybridization of RNA to a probe specific for the gene, and this RNA directed the synthesis in vitro of TK polypeptides. Deletion of the 5' end of the gene seems to affect the stability of either the enzyme or TK-specific mRNA, or both. The TMP phosphorylating activity seems to be particularly destabilized relative to the thymidine phosphorylating activity.
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PMID:Evidence that deletion of coding sequences in the 5' end of the thymidine kinase gene of herpes simplex virus type 1 affects the stability of the gene products. 282 63

The genome of herpes simplex virus codes for several enzymes, including viral thymidine kinase and viral deoxyribonucleic acid (DNA) polymerase. When viral resistance develops, it does so by changes in these two enzymes. Three possible mechanisms of viral resistance to acyclovir include (1) selection of viral mutants that make little or no thymidine kinase and do not phosphorylate acyclovir adequately, (2) selection of mutants that can phosphorylate thymidine but cannot phosphorylate acyclovir (i.e., these viruses have thymidine kinases with altered substrate specificity), and (3) selection of viruses that have altered DNA polymerases that replicate viral DNA in the presence of acyclovir triphosphate. Thymidine kinase-deficient virus has been isolated from clinical isolates frequently, but few strains appear to be virulent for animals or humans and only a few seem to have caused clinical disease. Viruses with altered substrate specificity have been reported but viruses with an altered DNA polymerase have not occurred in clinical practice. Antiviral drugs should be used only when necessary to minimize the appearance of resistant strains of virus.
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PMID:Significance of resistance of herpes simplex virus to acyclovir. 282 41

Thymidine kinase from herpes simplex virus type 1 (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21) has been purified from an overexpression system and crystallized against ammonium sulfate by using the hanging-drop technique. The tetragonal crystals are of space group P4122 or P4322, and have unit cell dimensions a = b = 84 A, c = 180 A.
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PMID:Purification and crystallization of thymidine kinase from herpes simplex virus type 1. 284 4

Dietary fibers may tend to enhance or inhibit chemically induced experimental colon cancer, depending on the particular fiber consumed. This study examined the relationship between colonic thymidine kinase enzyme activity and mucin histochemistry and the reported effects of various dietary fibers on chemically induced colon carcinogenesis. Fiber-supplemented diets containing fibers reported to inhibit (wheat bran) or enhance (guar gum, carrageenan) chemically induced colon carcinogenesis in the rat were selected. Four groups of male Fischer 344 rats consumed 10% wheat bran, 5% guar gum, 5% carrageenan, or fiber-free diets ad libitum for 4 weeks. At the completion of the treatment period, the distal 12 cm of colonic mucosa was scraped off and homogenized for determination of thymidine kinase activity, and a 0.5-cm section of midcolon was processed by the high-iron diamine/Alcian blue method for mucin histochemistry. Final animal weights did not differ significantly among groups. Thymidine kinase enzyme specific activity (mumole thymidine phosphate formed x 10(6)/min/mg protein, means +/- SEMs) was not significantly different in the fiber-free, wheat bran, and guar gum groups (10.98 +/- 1.50, 7.41 +/- 1.09, and 9.11 +/- 2.04, respectively) but was markedly elevated at 41.84 +/- 4.65 in the carrageenan group (alpha less than 0.001). Mucin histochemistry failed to reveal any significant differences among dietary groups.
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PMID:Alterations in colonic thymidine kinase enzyme activity induced by consumption of various dietary fibers. 284 79

Antiviral activities of five nucleoside analogs against the VR-3 and WT-34 strains of herpes simplex virus type 1 (HSV-1) were investigated in Vero and human embryo lung fibroblast (HEL) cells. In HEL cells, the compounds showed antiviral activities against both strains of HSV-1, but in Vero cells, the antiviral activities of the compounds were reduced in proportion to their antiviral indexes (the 50% inhibitory dose [ID50] for cell growth divided by the 50% plaque reduction dose for virus). The ratio of the ID50 in Vero cells to the ID50 in HEL cells was larger in VR-3-infected cells than in WT-34-infected cells. The following results were obtained. (i) Thymidine kinase (TK; EC 2.7.1.21) activity in the VR-3- or WT-34-infected Vero cells was about half that in VR-3- or WT-34-infected HEL cells. Induction of viral TK was especially low in the VR-3-infected Vero cells. (ii) The ID50 of the plaque reduction assay in hypoxanthine, aminopterin, and thymidine medium revealed that the activity of cellular thymidylate synthetase (EC 2.1.1.45) was important in viral replication in VR-3-infected Vero cells. (iii) The VR-3-infected cells required larger thymidine and thymidine phosphate pools for viral replication than the WT-34-infected cells did, although uptake of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil into infected cells was equal for both strains. (iv) In the VR-3-infected Vero cells, the quantity of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil triphosphate was smaller than that in VR-3-infected HEL cells and WT-34-infected Vero and HEL cells.
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PMID:Efficacies of antiherpesvirus nucleosides against two strains of herpes simplex virus type 1 in Vero and human embryo lung fibroblast cells. 284 37

Analyses of enzymes from various metabolic pathways in pulmonary carcinoid tumors and radiological measurements of their volume increase were compared with those for lung carcinomas of various cell types. The results describe new biochemical features in carcinoid tumors, present the first quantitative evidence for their slow growth rate (i.e., long doubling time) in vivo, and show that measurement of 2 or 3 appropriate enzymes in biopsy samples can guard against instances in which carcinoids and adeno- or oat cell carcinomas are mistaken for one another on histological examination. The uridine kinase to thymidine kinase ratio as well as the beta-galactosidase concentration of carcinoid tumors were 5 times higher than of carcinomas, and their gamma-glutamyl transpeptidase was below that of all 35 adeno- and the 11 squamous cell carcinomas. Thymidine kinase, which bears a quantitative inverse correlation to volume doubling time (irrespective of cell type), had much lower titers in the 9 carcinoids than in the 6 oat cell carcinomas and reflects most clearly their very different (despite common histogenesis) clinical malignancy. Owing to their long doubling time, carcinoid tumors on the average required a much longer period (40.5 years) to attain final volume than did carcinomas (17.8 years). The calculated mean age of the subjects when growth began, -0.5 years (as opposed to 51 years for carcinomas), suggests a prenatal or early childhood inception for pulmonary carcinoid tumors.
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PMID:Pulmonary carcinoid tumors: enzymic discriminants, growth rate, and early age of inception. 287 Jul 99

The thymidine kinase gene of herpes simplex virus 1 was mutated by inserting oligodeoxynucleotide linkers into the region of the gene corresponding to the 5' untranslated portion of the mRNA. These linkers, when transcribed into mRNA, might be expected to form hairpin loops and hence to increase the secondary structure of the 5' end of the mRNA. Thymidine kinase insertion derivatives were examined in vivo and in vitro to determine translational efficiency. For the in vivo studies, thymidine kinase insertion derivatives were transfected into thymidine kinase deficient L cells alone and together with a selectable dominant marker, or were assayed in the COS-1 transient expression system. For in vitro studies, thymidine kinase insertion derivatives were subcloned into pSP64. Capped transcripts were analyzed for their ability to bind ribosomes and translate in rabbit reticulocyte lysates and wheat-germ extracts. The results demonstrate that translation efficiency is decreased as the number of linkers is increased and support the view that excessive secondary structure at the 5' end of eukaryotic mRNA impedes translation.
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PMID:Insertion mutagenesis to increase secondary structure within the 5' noncoding region of a eukaryotic mRNA reduces translational efficiency. 298 96

Thymidine kinase negative mouse (LATK-) and hamster (BHKTK-) cells were transfected with a recombinant plasmid (pTK1) carrying the thymidine kinase gene of HSV-1 by iontophoretic pricking or iontophoretic microinjection techniques. Transfection frequencies were measured using short term survival and long term transformation assays and were found to be at the level of 10 to 15% and 1 to 2% respectively for mouse or hamster cells transfected with pTK1. The presence of covalently linked transcriptional enhancers from SV40 or Moloney MSV increased long term transformation frequencies approximately 10-fold. Transfection by iontophoretic pricking of non-tumorigenic mouse (NIH3T3) or hamster (BHKC13) cells with a recombinant (pAGT1) carrying the human T24 Ha-ras1 oncogene and the bacterial aminoglycoside phosphotransferase (aph) gene as a selectable marker resulted in transformation of these cells. Southern blot hybridization analyses demonstrated the presence of circular, integrated or rearranged donor DNA molecules in the transformed cells. These gene transfer techniques and iontophoretic pricking in particular should be useful for the transfection of a variety of cell lines and markers.
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PMID:Transformation of mammalian cell by iontophoretic pricking or iontophoretic microinjection. 299 79

The herpes simplex virus (HSV) type 1 dUTPase gene was inactivated by insertion of HindIII oligonucleotide linker sequences into the KpnI site within the coding region of the cloned gene. The mutated gene was introduced into wild type herpes simplex virus by marker rescue and the recombinants were identified by the acquisition of a HindIII site within genome map coordinates 0.69 to 0.70 and the failure to induce virus-specific dUTPase activity. A spontaneous dUTPase deficient mutant, which had an identical restriction endonuclease DNA pattern to wild type virus, was also isolated from this transfection experiment. Both types of dUTPase-negative mutants failed to induce a virus-specific 39,000 mol wt polypeptide. Cells infected with the insertional mutant contained instead a novel polypeptide about 40,000 mol wt. No abnormal virus specific polypeptide was detected in cells infected with the spontaneous mutant. We conclude that the 39,000 mol wt polypeptide induced by wild type HSV-1 is the virus-coded dUTPase. Since both types of mutants grew well in exponentially growing and serum-starved tissue culture cells in the absence of wild type helper virus, the dUTPase is not required for virus replication under these conditions. Thymidine kinase deficient, dUTPase deficient double mutants were constructed by recombination of a thymidine kinase insertional mutation into dUTPase deficient virus. These mutants also grew as well as wild type virus both in normal tissue culture cells and cells lacking the cellular thymidine kinase.
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PMID:Isolation and characterisation of herpes simplex virus type 1 mutants which fail to induce dUTPase activity. 300 29


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