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)

Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.
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PMID:Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26. 789 98

A human cDNA sequence homologous to human deoxycytidine kinase (dCK; EC 2.7.1.74) was identified in the GenBank sequence data base. The longest open reading frame encoded a protein that was 48% identical to dCK at the amino acid level. The cDNA was expressed in Escherichia coli and shown to encode a protein with the same substrate specificity as described for the mitochondrial deoxyguanosine kinase (dGK; EC 2.7.1.113). The N terminus of the deduced amino acid sequence had properties characteristic for a mitochondrial translocation signal, and cleavage at a putative mitochondrial peptidase cleavage site would give a mature protein size of 28 kDa. Northern blot analysis determined the length of dGK mRNA to 1.3 kbp with no cross-hybridization to the 2.8-kbp dCK mRNA. dGK mRNA was detected in all tissues investigated with the highest expression levels in muscle, brain, liver, and lymphoid tissues. Alignment of the dGK and herpes simplex virus type 1 thymidine kinase amino acid sequences showed that five regions, including the substrate-binding pocket and the ATP-binding glycine loop, were also conserved in dGK. To our knowledge, this is the first report of a cloned mitochondrial nucleoside kinase and the first demonstration of a general sequence homology between two mammalian deoxyribonucleoside kinases. Our findings suggest that dCK and dGK are evolutionarily related, as well as related to the family of herpes virus thymidine kinases.
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PMID:Cloning and expression of human deoxyguanosine kinase cDNA. 869 79

In Lactobacillus acidophilus R-26, the synthesis of DNA precursor deoxynucleotides occurs exclusively by salvage of deoxynucleosides, beginning with phosphorylation by four deoxynucleoside kinases. Subunits bearing three of these activities are uniquely organized into two heterodimers, deoxyadenosine/deoxycytidine kinase (dAK/dCK) and deoxyadenosine/deoxyguanosine kinase (dAK/dGK), which, along with a distinct deoxythymidine kinase (TK), catalyze the parallel first committed steps of dNTP biosynthesis. Whereas TK is common to most prokaryotes (and eukaryotes), the other three activities that are the emphasis of this review are quite unusual in bacteria. Each activity is regulated in cis by its homologous end-product (dNTP) which is understood to act as a multisubstrate inhibitor capable of binding to both nucleoside and phosphate subsites. Conversely, the inactive dAK subunit is progressively activated by 1) association with a dGK or dCK subunit and 2) the conformationally driven heterotropic affect of dGuo or dCyd bound to the opposing subunit. Limited proteolysis has proven to be a powerful probe of conformational states. Further indication of conformational or structural differences between dAK and dGK (or dCK) is that the former follows an ordered kinetic path, while dGK or dCK exhibits rapid-equilibrium random kinetics. The multi-substrate behavior of end-product binding provides a convenient new diagnostic tool for distinguishing kinetic mechanisms. Tandem dak-dgk genes have been cloned from Lactobacillus DNA and expressed in Escherichia coli as dAK/dGK, utilizing the associated promoter. Sequence alignments reveal 65% identity in their DNA and 61% in their derived amino acid sequences. Encoded N-terminal sequences are identical for the first 18 residues, and both subunits share conserved sequences in common with adenylate kinase and viral TK. A more unusual conserved element, which appears to play a role in the activation of dAK, resembles the G2 loop of p21 ras. Remarkably, no homologous gene(s) for the dAK/dCK pair could be found. Comparisons of amino acid sequences, isoelectric pHs and subunit masses strongly indicated that native dCK and dGK are identical in sequence, except at their extreme N-termini (M-IVL for dCK and -TVIVL for dGK), suggesting that processing of a common precursor occurs in Lactobacillus. Accordingly, deletion of codons 2 and 3 from dgk resulted in the expression of dAK/dCK in the E. coli host; its kinetic properties are indistinguishable from those of native dAK/dCK. Subcloning the dgk or engineered dck gene resulted in expression of active dGK or dCK homodimers, each with a virtually unchanged Km toward its primary deoxynucleoside. However, in common with human dCK, dCK (or dGK) homodimer exhibits secondary activities with much larger Kms towards dAdo and dGuo (or dCyd). dCTP (or dGTP) is the best inhibitor of all three activities of the respective homodimer. Fully active heterodimers can be reconstituted simply by mixing a homodimer with independently expressed (inactive) dAK.
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PMID:Life on the salvage path: the deoxynucleoside kinase of Lactobacillus acidophilus R-26. 942 44

In mammalian cells, there are three pyrimidine nucleoside salvage enzymes with the capacity to phosphorylate all four deoxynucleosides, the two thymidine kinase isoenzymes, TK1 and TK2, and the deoxycytidine kinase, dCK. TK1 is cell cycle-regulated; TK2 is expressed constitutively and can phosphorylate deoxycytidine to the same extent as thymidine. dCK phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, but not thymidine. In addition, the three kinases can phosphorylate a number of medically important analogs. In cultured Drosophila melanogaster embryonic cells, only one pyrimidine deoxynucleoside kinase was present. This kinase was purified and showed a broad substrate specificity, since it was able to phosphorylate all four deoxynucleosides with high efficiency, as compared with the kinases in mammalian cells. Additionally, a number of nucleoside analogs such as arabinofuranosyl pyrimidines, deoxyuridine, and 5'-fluorodeoxyuridine, were phosphorylated. There was negligible 3'-azidothymidine and no dTMP phosphorylation. The enzyme was active as a monomer of about 30 kDa. We suggest the name D. melanogaster deoxynucleoside kinase for this multifunctional kinase. The substrate specificity, size, and other characteristics show that this enzyme is more related to human TK2 than to the other mammalian deoxyribonucleoside kinases, but is unique with respect to the capacity to phosphorylate all four deoxynucleosides.
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PMID:Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase. 946 77

Deoxycytidine kinase was shown to be activated during 2-chlorodeoxyadenosine (CdA) treatment of human lymphocytes, under the conditions when the DNA synthesis is inhibited. As the increase of dCK activity was shown in crude protein extracts, without an increase in the amount of dCK protein, shown by immunostaining after SDS-PAGE, a secondary modification of the protein structure was considered. NaF treatment of cells in the concentration range of 5-20 mM gave a similar activation of dCK, suggesting a possible role of phosphatases and/or a possibility of a G-protein related phenomenon. Using the same conditions, no effect of CdA or NaF was found on the thymidine kinase activity of cell extracts. Alternatively, activation of catabolic pathways could be considered, however, the increase in dCK activity was not influenced either by the removal of 5'-nucleotidases, or by the inhibition of deaminases.
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PMID:Activation of deoxycytidine kinase during inhibition of DNA synthesis in human lymphocytes. 959 21

To develop a suitable suicide gene/prodrug therapy for the treatment of thyroid carcinomas, the relative therapeutic efficacy of four different suicide gene/prodrug combinations was compared in thyroid carcinomas in vitro. Herpes simplex virus thymidine kinase and ganciclovir (HSV-TK/GCV), Escherichia coli cytosine deaminase and 5-fluorocytosine (CD/5FC), E coli nitroreductase and CB1954 (NTR/CB1954), and human deoxycytidine kinase and cytosine arabinoside (dCK/AraC) were employed. The suicide genes were transduced into two thyroid carcinoma cell lines with retroviral vectors in which all the suicide genes were under the control of the same promoter. When the relative efficacy of four suicide gene/prodrugs was compared with therapeutic index and degree of bystander effect, we found a clear dissociation between these two parameters. Thus, HSV-TKIGCV demonstrated the widest therapeutic index, while CD/5FC and NTR/CB1954 showed the stronger bystander effect than HSV-TK/GCV. dCK/AraC had little efficacy. Advantages and limitations of each suicide gene/prodrug combinations are discussed.
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PMID:Treatment of thyroid carcinoma cells with four different suicide gene/prodrug combinations in vitro. 967 64

OBJECTIVES: In vitro activities of thymidine kinase (TK, EC 2.7.1.21), adenosine kinase (AK, EC 2.7.1.20) and deoxycytidine kinase (dCK, EC 2.7.1.74) enzymes involved in the salvage pathway of DNA precursor synthesis, in homogenates of the rat liver and kidney, were examined. Type I iodothyronine-5'-deiodinase (5'D-I) is the main enzyme responsible for peripheral metabolism of thyroid hormones. This occurs especially in the liver, kidney and muscle. The activity of 5'D-I is inhibited bypropylthiouracil (PTU), an antithyroid drug. METHODS: The liver and kidney were collected from rats pretreated in vivo with either a 0.1% solution of PTU in drinking water for 2 weeks or injected with levothyroxine (L-T(4), 50 &mgr;g/kg BW, daily) for 2 weeks. The enzyme activities were measured by ascending chromatography and expressed asthe amounts of radioactive reaction products of the phosphorylation of dThd (for TK), ofdAdo (for AK and dCK) and of dGuo (for dCK). RESULTS: In liver homogenates, PTU-pretreatment decreased the activities of the three enzymes when compared to control values and those of L-T(4)-treated animals; also L-T(4) injections decreased the AK and dCK activities in the liver homogenates. PTU-pretreatment increased TK activity and the rate of dGuo phosphorylation in kidney homogenates, when compared to controls and to the L-T(4)-pretreated animals. Conversely, both PTU- and L-T(4)-pretreatment reduced the rate of dAdo phosphorylation in kidney homogenates. CONCLUSION: Changes in the activities of examined enzymes which participate inpyrimidine orpurine metabolism of the salvage pathway of DNA synthesis in the liver afterPTU-pretreatment (as shown herein) are similar to the changes of the 5'D-I activity after PTU-treatment (as reported by others). Thus, the observations suggest a role of the salvage pathway of DNA synthesis in the peripheral metabolism of thyroid hormones.
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PMID:Pyrimidine and purine salvage deoxyribonucleoside metabolism in hepatic and renal homogenates from rats pretreated with propylthiouracil or L-thyroxine. 1145 32

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.
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PMID:Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation. 1181 27

Increased activities of some enzymes, which participate in pyrimidine and purine salvage pathway, were found in blood fractions of patients suffering from different autoimmunological diseases, thyroid diseases included. The aim of the study was to estimate the expression of genes, specific for deoxycytidine kinase (dCK, EC 3.7.1.74), thymidine kinase 1 (TK1; EC 2.7.1.21), and adenosine deaminase (ADA, EC 3.5.4.4) in blood leukocytes, collected from patients with autoimmunological thyroid diseases (AITD), i.e., Graves' or Hashimoto's disease. The total mRNA was isolated from peripheral blood leukocytes and, afterwards, submitted to reverse transcription (RT), with the following amplification of genes encoding for particular examined enzymes and beta-actin, as a supervisory gene [RT-polymerase chain reaction (RT-PCR)]; ADA gene was amplified with the use of three different primer pairs (ADA3, ADA4, and ADA5). PCR products were electrophoresed in 8% polyacrylamide gel and then, submitted to densitometric analysis. The levels of expression of all the examined genes in leukocytes from patients with either Graves' or Hashimoto's disease were significantly increased when compared to those in controls; above a twofold elevation of expression of TK1, ADA4, and ADA5 genes was observed. In conclusion, the changes of activities of salvage enzymes in patients with AITD occur likely at transcription level; the measurement of gene expression for purine and pyrimidyne salvage enzymes may likely help explain the mechanism of autoimmune diseases, being also significant in the diagnostics and/or monitoring of AITD.
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PMID:Expression of genes for certain enzymes of pyrimidine and purine salvage pathway in peripheral blood leukocytes collected from patients with Graves' or Hashimoto's disease. 1276 88

Anti-HIV nucleoside therapy can result in mitochondrial toxicity affecting muscles, peripheral nerves, pancreas and adipose tissue. The cytosolic deoxycytidine kinase (dCK; EC 2.7.1.74) and thymidine kinase (TK1; EC 2.7.1.21), the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK; EC 2.7.1.113) as well as 5'-deoxynucleotidases (5'-dNT; EC 3.1.3.5) are enzymes that control rate-limiting steps in formation of intracellular and intra-mitochondrial nucleotides. The mRNA levels and activities of these enzymes were determined in mouse tissues, using real-time PCR and selective enzyme assays. The expression of mRNA for all these enzymes and the mitochondrial deoxynucleotide carrier was detected in all tissues with a 5-10-fold variation. TK1 activities were only clearly detected in spleen and testis, while TK2, dGK and dCK activities were found in all tissues. dGK activities were higher than any other dNK in all tissues, except spleen and testis. In skeletal muscle dGK activity was 5-fold lower, TK2 and dCK levels were 10-fold lower as compared with other tissues. The variation in 5'-dNT activities was about eight-fold with the highest levels in brain and lowest in brown fat. Thus, the salvage of deoxynucleosides in muscles is 5-10-fold lower as compared to other non-proliferating tissues and 100-fold lower compared to spleen. These results may help to explain tissue specific toxicity observed with nucleoside analogs used in HIV treatment as well as symptoms in inherited mitochondrial TK2 deficiencies.
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PMID:Expression of deoxynucleoside kinases and 5'-nucleotidases in mouse tissues: implications for mitochondrial toxicity. 1749 87


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