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)

In mammalian cells, salvage pathway phosphorylation of thymidine is catalyzed by two thymidine kinases: the cell-cycle regulated cytoplasmic TK1 and the constitutively expressed mitochondrial TK2. Since TK1 is virtually absent in non-dividing cells, TK2 is probably the only thymidine kinase present in these cells. In cellular metabolism, TK1 and TK2 presumably serve to maintain sufficient dTTP for DNA replication and repair. TK1 purified from phytohemagglutinin-stimulated human lymphocytes is a dimer in the absence and a tetramer in the presence of ATP. In addition to the molecular weight transition, incubation with ATP at 4 degrees C or storage with ATP induces a reversible, enzyme concentration-dependent, kinetically slow transition from a low to a high affinity form of TK1, with Km values of 14 microM and 0.5 microM, respectively. This affinity difference implies that at cellular thymidine concentrations, the difference in catalytic activity between the two TK1 forms will be 3-5-fold. Calculations of cellular TK1 concentration suggested that the low affinity dimer form was dominant in G0/G1 cells and the high affinity tetramer form in S-phase cells. Hence, the transition may serve to fine-tune the cell-cycle regulation of thymidine kinase activity on the post-translational level. To study the ATP effect on the molecular level, an IPTG inducible T7 RNA polymerase-dependent expression system for the entire human TK1 polypeptide in E. coli was established. The recombinant TK1 has the same subunit mass and specific activity as the native enzyme. However, the recombinant TK1 solely displayed the kinetics of the high affinity form, with Km values of 0.3-0.4 microM regardless of pre-exposure to ATP, indicating that the ATP effect may be dependent on post-translational modifications absent in E. coli. Surprisingly, we did not observe any effect of ATP on TK1 purified from bone-marrow cells from a patient with acute monocytic leukemia (AMOL). Furthermore, the Km values of TK1 from these cells were 45 microM for the ATP-free enzyme and 65 microM for the ATP-incubated enzyme. With TK1 purified from HL-60 cells, we obtained the same pattern and kinetic values as for TK1 from lymphocytes. In the light of the results with the recombinant TK1, we presume that the lack of ATP effect and very high Km values observed for the AMOL TK1 may be due to changes in post-translational regulatory mechanisms in acute monocytic cells.
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PMID:Human thymidine kinase 1. Regulation in normal and malignant cells. 757 55

Recent results showed that ATP enables a kinetically slow shift from a low affinity form to a high affinity form of human cytosolic thymidine kinase (TK1), as reflected by the respective apparent Km values for thymidine of 15 microM and 0.7 microM. The shift is dependent on the concentration of enzyme protein, and calculations indicate that the low affinity form is predominant in G1 cells, and the high affinity form is predominant in S-phase cells. Here, we report that the two forms of TK1 differ manyfold in affinity to the substrate ATP, to the inhibitor dTTP and to various analogs of thymidine substituted in the pyrimidine or sugar. Furthermore, the kinetic reaction mechanisms suggest that the nucleoside analog. 3'-azidothymidine, used for treatment of infections with human immune deficiency virus (HIV), is not a substrate for the low affinity form of TK1.
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PMID:Different affinity of the two forms of human cytosolic thymidine kinase towards pyrimidine analogs. 763 20

Deoxynucleoside kinases are key enzymes in deoxyribonucleoside salvage, activating several clinically important chemotherapeutic drugs. The four known kinases, cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK), have been purified and characterized as to the subunit structure as well as specificity with a large number of analogs. These results are summarized and used to establish selective assays for the four enzymes in crude extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas. TK2 and dGK activities were found at low levels in all tissues, possibly correlated to the content of mitochondria. TK1 activity was detected only in samples containing a significant number of S phase cells. We have measured dCK activity as well as dCK polypeptide level by immuno blotting in these extracts. High levels of dCK were found in normal mononuclear leukocytes (91-145 ng dCK/mg protein) and in B-cell chronic lymphocytic leukemia (80 +/- 30 ng/mg, n = 23). Hairy cell leukemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did unexpectedly three samples of T-cell chronic lymphocytic leukemia (18 +/- 14 ng/mg). Phytohemaglutinine stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or expression (less than 2.5-fold). In colon adenocarcinomas, the dCK content was significantly higher (21 +/- 9.3 ng/mg, n = 20) than in normal colon mucosa (8.2 +/- 3.7 ng/mg, n = 19, p < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal ventricular mucosa (6.2 +/- 5.4 ng/mg, n = 5, p < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed a dCK levels comparable to those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg), while other sarcoma samples contained levels comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). We confirm that dCK is expressed constitutively and predominantly in lymphoid cells, but conclude that a significant expression may be found in non-lymphoid tissues as well, with increased levels in the corresponding tumor tissue. 2-Chlorodeoxyadenosine (CdA), an antileukemic agent used in treatment of hairy cell leukemia and chronic lymphocytic leukemias (B-CLL), is phosphorylated by dCK which was used as the selective substrate for this enzyme. A study was performed to investigate if there was a correlation between the dCK levels and the response to CdA treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy. 794 71

The level of cytosolic thymidine kinase (TK1) mRNA in lymphocytes from six healthy people and in lymphocytes from five patients with untreated chronic lymphatic leukemia (CLL) was determined with competitive polymerase chain reaction (competitive PCR). Using this procedure we have shown that in patients with CLL, there is an overexpression of TK1 mRNA without corresponding enzymatic activity. The TK1 mRNA level is approximately 100-fold higher in lymphocytes from CLL patients than in lymphocytes from healthy persons. A high level of TK1 mRNA without corresponding enzyme activity may indicate a defect in the processing of the enzyme. This may disturb the cells' normal feedback system and thereby influence the development of malignant conditions.
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PMID:Overexpression of human thymidine kinase mRNA without corresponding enzymatic activity in patients with chronic lymphatic leukemia. 796 13

We report here the assignment of TK1, the gene for thymidine kinase to Syrian hamster (Mesocricetus auratus) chromosome 9 (MAU9) by complementation mapping. Syrian hamster chromosomes derived from a wild type (TK+) subline of BHK cells were introduced via microcell-mediated chromosome transfer into B82 mouse cells deficient in thymidine kinase (TK-), a defect that prevents their growth in HAT culture media. Hybrid clones were selected in HAT media. Chromosome analyses of the microcell hybrids showed that the thymidine kinase deficiency of B82 cells was corrected by MAU9. Therefore, we assigned TK1 to MAU9. Previously, TK1 was assigned to mouse chromosome 11 (MMU11), rat chromosome 10 (RNO10), Chinese hamster chromosome 7 (CGR7), and human chromosome 17 (HSA17). The striking banding homology of MAU9 with RNO10, MMU11, CGR7 and HSA17 provides additional support for the assignment of TK1 to MAU9. To our knowledge, this is the first report of gene assignment to a specific Syrian hamster chromosome using the somatic cell hybridization technique.
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PMID:Assignment of TK1 encoding thymidine kinase to Syrian hamster chromosome 9 by microcell-mediated chromosome transfer. 812 17

Human cytosolic thymidine kinase, subunit molecular mass about 24 kDa, is a tetramer in the presence of ATP but a dimer in the presence of thymidine or without substrates. The pure, substrate-free enzyme showed complex, non-hyperbolic thymidine substrate kinetics with an apparent Km of 15 microM. Incubation with ATP at 4 degrees C induced a time-dependent transition to an enzyme form with hyperbolic kinetics and a 20-fold lower Km value for thymidine (0.7 microM) but the same maximal velocity as for cytosolic thymidine kinase (TK1) without ATP. Removal of the ATP by carboxymethyl chromatography reestablished the non-hyperbolic kinetics with the low affinity for thymidine (Km(app) = 12 microM), and this enzyme form could be reversed once more by ATP incubation to the high affinity enzyme form. Similar shifts could not be induced by thymidine. The activating effect of ATP depended on the concentration of enzyme protein in a linear manner. These results indicate that ATP is a positive effector of cytosolic thymidine kinase, controlling a kinetically slow transition between two molecular forms of the enzyme. A hypothetical reaction mechanism is presented to explain the complex kinetic behavior.
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PMID:Reversible ATP-dependent transition between two forms of human cytosolic thymidine kinase with different enzymatic properties. 834 Mar 87

We have mapped the autosomal sex reversal locus, SRA1, associated with campomelic dysplasia (CMPD1) to 17q24.3-q25.1 by three independent apparently balanced de novo reciprocal translocations. Chromosome painting indicates that the translocated segment of 17q involves about 15% of chromosome 17 in all three translocations, corresponding to a breakpoint at the interphase between 17q24-q25. All three 17q breakpoints were localized distal to the growth hormone locus (GH), and proximal to thymidine kinase (TK1). Due to the distal location of the breakpoints, previously mentioned candidate genes, HOX2 and COL1A1, can be excluded as being involved in CMPD1/SRA1. The mouse mutant tail-short (Ts) which maps to the homologous syntenic region on mouse chromosome 11, displays some of the features of CMPD1.
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PMID:Assignment of an autosomal sex reversal locus (SRA1) and campomelic dysplasia (CMPD1) to 17q24.3-q25.1. 834 55

The majority of primary bladder neoplasms are known to arise within the mucosa around the ureteric orifices and bladder base. This may be due to the mucosa in this area being more susceptible to carcinogens than other areas of the bladder. Deficiency in the nucleotide salvage pathway enzyme thymidine kinase (TK), and especially its TK1 isozyme, has been shown to predispose cell lines to increased mutagenesis. Total TK and TK1 activities were measured in mucosal samples taken adjacent to the ureteric orifices and dome in 32 normal bladders and both total TK and TK1 were shown to be significantly decreased in the mucosa adjacent to the ureteric orifices. This may explain why primary bladder neoplasms occur more commonly in this site.
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PMID:Why do most primary bladder neoplasms first appear around the ureteric orifices? 843 34

Brain tumors have been treated clinically by intratumoral injection of cells that produce retroviral vectors encoding the herpes simplex virus thymidine kinase (HSV-TK) gene followed by systemic administration of the antiviral drug ganciclovir. In vitro and in vivo comparisons of two different HSV-TK vector producer clones, which were made using standard transfection and transinfection techniques, were conducted. The two clones, PA317/G1TkSvNa.53 (TK.53) and PA317/G1Tk1SvNa.7 (TK1.7), both used in clinical trials, differ with respect to sequences 3' to the HSV-TK stop codon. The retroviral construct used to generate the TK.53 vector producer cell clone contains an open reading frame encoding a portion of the herpes simplex virus glycoprotein H (gH), a potential polyadenylation site and a putative splice site in this region. These sequences were removed from the retroviral construct used to create the TK1.7 vector producer cell clone. Supernatants obtained from TK1.7 vector producer cells had 100- to 1000-fold higher titers (G418 or HAT) than did corresponding supernatants from TK.53 vector producer cells. A murine subcutaneous tumor model was used to assess transduction efficiency and antitumor activity of each vector producer cell clone. In vivo tumor cell transduction was 13- to 18-fold more efficient with TK1.7 cells as compared with TK.53 cells at equivalent doses. Complete tumor ablation was achieved using a 10-fold lower dose of TK1.7 cells as compared with TK.53 cells. These results suggest that TK1.7 cells combined with ganciclovir may provide a more potent antitumor response in humans.
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PMID:An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases antitumor efficacy in vivo. 854 81

Soluble thymidine kinase (TK1) is an important 17q marker in somatic cell genetics. Its activity is increased in many malignancies, including breast cancer. Through somatic cell hybrid and fluorescence in situ hybridization studies, we mapped TK1 to 17q25.2-->25.3, in region demonstrating loss of heterozygosity in primary breast tumors. It lies near D17S836 and is proximal to the avian erythroblastic leukemia viral oncogene homolog 2-like gene (ERBA2L).
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PMID:FISH localization of the soluble thymidine kinase gene (TK1) to human 17q25, a region of chromosomal loss in sporadic breast tumors. 864 Nov 39


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