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)

Thymidine kinase 2 (TK2), also called mitochondrial thymidine kinase, is a pyrimidine deoxyribonucleoside kinase expressed in all cells and tissues. It was recently purified to apparent homogeneity from human leukemic spleen and the active enzyme was shown to be a monomer of a 29-kDa polypeptide. The enzyme is feedback-inhibited by both end products, dCTP and dTTP. Here we show that TK2 purified from several different sources, including purified beef heart mitochondria, could be directly photoaffinity labeled with radioactive dTTP (approximately 18% of all TK2 molecules were cross-linked to dTTP after 20 min of ultraviolet irradiation) or to a lower extent with dCTP. Photo-incorporation was inhibited by the presence of the other effector but also the phosphate donor ATP blocked photolabeling, with dTTP. Addition of nucleoside substrates gave only a marginal inhibition of photo-incorporation. There were no detectable difference in the molecular size of photolabeled TK2 isolated from human spleen, brain or placenta, monkey liver, beef heart and beef heart mitochondria. Nor was there any significant differences in the enzyme kinetic properties of these enzymes. Cleavage of labeled TK2 with cyanogen bromide showed that dTTP was incorporated into a single 3-kDa peptide. TK2 was the only pyrimidine deoxynucleoside kinase expressed in liver, heart and brain. A detailed characterization of the subunit structure and substrate specificity of this enzyme is of importance for the design of new antiviral and cytostatic therapies based on nucleoside analogs.
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PMID:Mammalian thymidine kinase 2. Direct photoaffinity labeling with [32P]dTTP of the enzyme from spleen, liver, heart and brain. 159 87

6-Methoxypurine arabinoside (ara-M) exhibits potent activity against varicella-zoster virus (VZV) as a result of ara-M's anabolism to the triphosphate of adenine arabinoside (ara-ATP) in VZV-infected cells. The adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) enhanced the formation of ara-ATP by inhibiting ara-M demethoxylation. In contrast, deoxycoformycin and coformycin, inhibitors of both adenosine deaminase and AMP deaminase, blocked the formation of ara-ATP and reversed the anti-VZV activity of ara-M. These results indicate that after the initial phosphorylation of ara-M by the VZV-coded thymidine kinase, the monophosphate is demethoxylated by AMP deaminase to form ara-IMP, which is converted to ara-ATP by the sequential actions of the cellular adenylosuccinate synthetase, adenylosuccinate lyase, and nucleotide kinases.
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PMID:Anabolic pathway of 6-methoxypurine arabinoside in cells infected with varicella-zoster virus. 166 24

An an initial step in the development of a recombinant poultry infectious laryngotracheitis virus (ILTV) vaccine, we report on the identification, cloning, and sequencing of a thymidine kinase (tk) gene from a virulent U.S. field isolate of ILTV. Degenerate oligonucleotide primers for the consensus nucleotide (ATP) binding site and the nucleoside (thymidine) binding site of other herpesvirus tk genes were used in the polymerase chain reaction (PCR) to amplify a fragment of ILTV DNA. The 344-base-pair (bp) amplified fragment was cloned into plasmid pKSII and used in Southern hybridizations to locate the ILTV tk gene on a 2.4-kb HindIII fragment. Upon cloning and sequencing this fragment, a 1089-bp open reading frame was identified, which is predicted to encode a protein demonstrating 27.9% amino acid homology to the herpes simplex virus type 1 (HSV-1) thymidine kinase protein. Analysis of the sequence revealed one region of difference from that reported for the Thorne strain of ILTV. In addition, the portion of the TK protein corresponding to the nucleotide binding domain is highly conserved among the avian herpesviruses.
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PMID:Identification of the thymidine kinase gene of infectious laryngotracheitis virus. 166 22

6-Methoxypurine arabinoside (ara-M) is a highly selective inhibitor of varicella-zoster virus (VZV). It belongs to a class of purine arabinosides whose anti-VZV activity in vitro correlates with substrate utilization by the VZV-encoded thymidine kinase (TK) (D. R. Averett, G. W. Koszalka, J. A. Fyfe, G. B. Roberts, D. J. M. Purifoy, and T. A. Krenitsky, Antimicrob Agents Chemother. 35:851-857, 1991). In this study, the mechanism of action of ara-M was explored. VZV-infected human fibroblasts selectively accumulated ara-M and its phosphorylated metabolites, whereas in uninfected fibroblasts or in those infected with a TK-deficient strain of VZV, there was virtually no cellular uptake of ara-M. The major intracellular metabolite of ara-M in VZV-infected cells was identified as the triphosphate of adenine arabinoside (ara-ATP). Appreciable levels of ara-ADP, ara-AMP, and ara-MMP were also detected. However, di- or triphosphorylated forms of ara-M were not detected. Moreover, in VZV-infected cells, the concentrations of ara-ATP which accumulated in the presence of ara-M were up to eightfold higher than those generated with ara-A itself. In contrast, in uninfected cells, the levels of ara-ATP which accumulated in the presence of ara-M were barely detectable. Clearly, Ara-M activation was dependent on the activity of the virus-encoded TK, while ara-A anabolism resulted primarily from the activity of host cell enzymes. Therefore, ara-M selectively generates the DNA polymerase inhibitor ara-ATP in the VZV-infected cell.
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PMID:Selective anabolism of 6-methoxypurine arabinoside in varicella-zoster virus-infected cells. 172 79

Activity of replicase complex enzymes involving thymidine kinase (TK), ribonucleotide reductase (RR), DNA-polymerases alpha and beta as well as DNA synthesis and single breaks in DNA were studied during growth of P388 ascites tumor. Under these conditions the rate of DNA synthesis was distinctly decreased via salvage pathway and de novo. Single breaks were not detected in the preexistent DNA within various periods after transplantation of P338 leukemic cells. Retardation of DNA synthesis during tumor growth correlated with a decrease in TK, RR and DNA-polymerase alpha activities, while DNA-polymerase beta activity was markedly increased. Growth of melanoma B16 was accompanied by a decrease in content of ATP, ADP, NAD, phosphocreatine and phosphosaccharides as well as by an increase in the level of inorganic phosphates.
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PMID:[Changes in the replication apparatus and phosphorus-containing metabolite pool in experimental tumors in animals during development]. 181 11

Alkylated nucleotides have been detected by 32P-postlabelling using the enzyme T4 polynucleotide kinase which phosphorylates the 3'-mononucleotides to give the 3',[5'-32P]bisphosphates. These may then be separated by two-dimensional TLC as the bisphosphates or the [5'-32P]monophosphates. We describe here an alternative approach using the Epstein-Barr virus (EBV) encoded thymidine kinase (TK) to directly phosphorylate adducted nucleosides to give the [5'-32P]monophosphates. Using a series of methyl, ethyl and butyl thymidines EBV-encoded TK was shown to phosphorylate a wide range of adducted thymidines with varying degrees of labelling efficiency; N3-methyl thymidine was labelled with the highest efficiency and O4-ethyl thymidine the lowest. Whereas O4-methyl thymidine was labelled at a higher efficiency than O2-methyl thymidine, O4-ethyl and O4-butyl thymidines were labelled at a much lower efficiency than the corresponding O2-alkyl thymidines. Labelling efficiency increased with pH in the range pH 7 to pH 9, but the relative labelling efficiency was ATP independent. This direct phosphorylation of adducted nucleosides offers an alternative approach to the detection of alkylated residues in DNA which may complement current postlabelling procedures.
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PMID:32P-postlabelling of alkylated thymidines using Epstein-Barr virus encoded thymidine kinase. 184 71

Thymidine kinase (EC 2.7.1.21) from regenerating rat liver has been purified 70,000-fold to apparent homogeneity by affinity chromatography. Molecular weight of the native enzyme was found to be about 54,000, as determined by gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate yielded a single band with a molecular weight of 26,000, suggesting that thymidine kinase is a dimer of very similar or identical subunits. The Michaelis constant for thymidine is 2.2 microM. ATP acts as a sigmoidal substrate with a 'Km' of 0.2 mM. Reaction kinetics and product inhibition studies reveal the enzymatic mechanism to be sequential.
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PMID:Purification and characterization of thymidine kinase from regenerating rat liver. 191 61

Thymidine kinase was isolated and purified 1600-fold from sea urchin (Strongylocentrotus intermedius) oocytes. The molecular mass of the enzyme is 66 kDa, pI is 5.2. The enzyme activity needs Mg2+, ATP and the corresponding phosphate acceptor. The pH optimum of the enzyme activity is at 9.0-9.5. The isolated enzyme does not exhibit any strict substrate specificity and can phosphorylate thymidine, deoxycytidine and some other pyrimidine nucleosides and their derivatives, the phosphorylation rate being maximal for thymidine, ATP or dATP. The TMP formed via the enzymatic reaction does not influence the thymidine kinase activity.
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PMID:[Thymidine kinase from sea urchin oocytes]. 196 86

(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

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.
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PMID:Biosynthesis and scavenging of pyrimidines by pathogenic mycobacteria. 219 Oct 77


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