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)

1. The hepatic metabolism of glutamine, alanine, ammonia, urea, glutathione and glucose was studied in rats made septic by caecal ligation and puncture and was compared with that in rats that had undergone sham operation (laparotomy). 2. Sepsis resulted in increases in the plasma activities of gamma-glutamyltransferase (P less than 0.001), alanine aminotransferase (P less than 0.001) and aspartate aminotransferase (P less than 0.001), the serum total and direct bilirubin concentrations (P less than 0.001), and the blood lactate (P less than 0.01), glutamine (P less than 0.05), alanine (P less than 0.001) and urea (P less than 0.05) concentrations, but produced decreases in the blood ketone body (P less than 0.001) and glutathione (P less than 0.05) concentrations and in the plasma cholesterol concentration (P less than 0.05). These changes were associated with marked negative nitrogen balance in septic rats. 3. Sepsis increased total hepatic blood flow (by 22.7%) together with hepatic arterial flow (by 25.8%) and portal venous flow (by 18.7%). Sepsis resulted in marked increases in the net rates of hepatic extraction of glutamine (by 164%), alanine (by 138%) and ammonia (by 259%) with concomitant increases in the net rates of hepatic release of glutamate (by 105%), glutathione (by 87.5%), glucose (by 70.1%) and urea (by 100.4%). 4. Sepsis increased the activities of liver carbamoylphosphate synthase (by 16.4%), ornithine transcarbamylase (by 29.8%), argininosuccinate synthase (by 28.1%) and arginase (by 33.8%). 5. Septic rats exhibited marked increases in hepatic protein (by 46.0%), RNA (by 43.4%) and DNA (by 37.7%) contents. These changes were accompanied by marked increases in the activity of thymidine kinase (by 35.9%).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hepatic glutamine metabolism in the septic rat. 137 98

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was found with 10(-8) M insulin. Insulin caused similar effects also on thymidine kinase activity. All these effects were obtained by using minimal essential medium without glutamine. The addition of glutamine to the medium reduced the inhibitory effect of insulin. Retinas of chick embryos contain immunoreactive insulin. Retinal immunoreactive insulin was at the highest level (1.12 ng/mg of protein) in the youngest retinas studied (day 6), then it declined with age, reaching the lowest value (0.58 ng/mg of protein) at day 14. This value did not vary significantly during the third week of development. A potential biological role of insulin in retinal development is discussed.
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PMID:Identification of insulin in chick embryo retina during development and its inhibitory effect on DNA synthesis. 154 69

Exploitation of differences in the substrate specificity of the type I and type II thymidine kinases (EC 2.7.1.21, TK) expressed by the Herpesviridae and Poxviridae (and human cells), respectively, has lead to the development of effective antiherpetic drugs such as acyclovir and gancyclovir. Analysis of type I TK protein sequences reveals a consensus sequence which corresponds to domain IV of type II TK proteins such as that encoded by vaccinia virus (VV). The type I descriptor (Xpho - + + Xpho) differs at the second position from the type II consensus sequence (Xpho Xphi + + Xpho) by having an aspartic acid residue (D) substituted for a glutamine (Q). To test the hypothesis that this substitution may be responsible for the observed differences in substrate specificity of these enzymes and as a approach to identify the nucleoside binding site of the type II VV TK, site-directed mutagenesis was employed to alter glutamine 114 (Q114) within domain IV of VV TK to a histidine (Q114H) or an aspartic acid (Q114D). All of the mutant enzymes retained full enzymatic activity as compared to wild-type VV TK when thymidine or bromodeoxyuridine were used as substrates. However, unlike the wild-type herpes simplex (type 1) TK enzyme, neither wild-type nor domain IV VV TK mutants were able to phosphorylate acyclovir or cytidine substrates. Surprisingly, the domain IV VVTK mutants displayed a dramatic loss of feedback inhibition by dTTP. Mutations of the Q114 position also lead to a difference in ATP binding as demonstrated by an altered elution pattern of Q114H and Q114D from an ATP-agarose affinity column with dTTP. Taken together, these results suggest that domain IV of VV TK is not involved directly in substrate discrimination but instead participates in feedback inhibition by dTTP.
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PMID:A single amino acid substitution abolishes feedback inhibition of vaccinia virus thymidine kinase. 157 11

Varicella-zoster virus (VZV) encodes a thymidine kinase (EC 2.7.2.21) which phosphorylates several antiviral nucleoside analogs, including acyclovir (ACV). A mutation in the VZV thymidine kinase coding sequence, resulting in an arginine-to-glutamine substitution at amino acid residue 130 (R130Q), is associated with clinical resistance to ACV. We have expressed the wild-type and the mutant enzymes in bacteria and have studied the kinetic characteristics of the purified enzymes. The arginine-to-glutamine substitution resulted in decreased catalytic activity and altered substrate specificity. The most striking effect was a decrease in the rates of nucleoside phosphorylation to less than 2% of the rates with the wild-type enzyme. This was accompanied by increased apparent Km values for thymidine and deoxycytidine. ACV was not detectably phosphorylated by the R130Q enzyme but still competed with thymidine for the enzyme. The inability of the R130Q enzyme to catalyze the phosphorylation of ACV correlates with resistance to ACV noted with a clinical isolate of VZV.
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PMID:Mutant varicella-zoster virus thymidine kinase: correlation of clinical resistance and enzyme impairment. 165 51

The isolation and description of acyclovir-resistant (ACVR) herpes simplex-2 viruses from patients with AIDS has recently been reported. These ACVR viruses were all markedly decreased in their thymidine kinase (TK) activity, and 6 of 10 of these TK viruses were able to establish latency. In addition, one of these isolates, ACVR-86012 was neuropathogenic in a murine encephalitis model. In this paper, the characteristics of these isolates with respect to TK polypeptide synthesis are examined. All but one isolate synthesized a detectable TK protein by immunoprecipitation, and 9/10 of the TK proteins had an altered electrophoretic mobility as compared to wild-type. The TK polypeptide from the neuropathogenic isolate ACVR-86012 was full-length and the gene was sequenced. An amino acid change from a glutamine to a proline at amino acid residue 105 was detected compared to the wild-type HSV-333 strain. These results indicate that an amino acid change in the NH2 portion of the TK protein is associated with a full-length peptide with decreased enzyme activity but the virus retains neuropathic virulence.
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PMID:Analysis of the thymidine kinase gene from clinically isolated acyclovir-resistant herpes simplex viruses. 184 99

The Sp1 protein activates transcription from many eukaryotic promoters. Sp1 can act in vivo from enhancer sites that are distal to the promoter and exhibit synergistic interaction with promoter-proximal binding sites. To investigate possible protein-protein interactions between DNA-bound Sp1 molecules, we have used electron microscopy to visualize the DNA-protein complexes. At the SV40 promoter, we observed the expected localized interaction at the Sp1 sites; in addition, we found that DNA-bound Sp1 served to associate two or more DNA molecules. At a modified thymidine kinase promoter, we observed a localized interaction at each of two binding locations that were separated by 1.8 kbp; in addition, we noted a substantial fraction of DNA molecules in which the distant binding regions were joined by a DNA loop. As judged by studies with mutant Sp1 proteins, the distant interactions depended on the glutamine-rich regions of Sp1 required for transcriptional activation. We conclude that DNA-bound Sp1 can self-associate, bringing together distant DNA segments. From the correlation between DNA looping in vitro and synergistic activation of the modified thymidine kinase promoter shown previously in vivo, we suggest that Sp1 exerts its transcriptional synergism by a direct protein-protein association that loops the intervening DNA. Our experiments support the DNA-looping model for the function of transcriptional enhancers.
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PMID:DNA looping between sites for transcriptional activation: self-association of DNA-bound Sp1. 185 Nov 21

The in vivo actions of two antimetabolites, acivicin (NSC-163501) and tiazofurin (NSC-286193), were examined on the enzymic programs of rat bone marrow. From the bone marrow of the femurs, 100,000 g supernatant fractions were prepared; enzymic activities were measured by isotopic assays, and cellularity was determined. In the normal bone marrow, the specific activities of pyrimidine de novo synthetic enzymes, CDP reductase, dTMP synthase, CTP synthase, carbamoyl-phosphate synthase II (synthase II), orotidine 5'-phosphate decarboxylase and aspartate carbamoyltransferase, were 1, 2.7, 5, 10, 63 and 601 nmol/hr/mg protein, respectively, whereas those of the salvage enzymes, deoxycytidine, thymidine, cytidine and uridine kinases were 3, 43, 149, and 367 nmol/hr/mg protein, respectively. In purine biosynthesis, the activities of the de novo synthetic enzymes, IMP dehydrogenase, formylglycinamidine ribonucleotide (FGAM) synthase, GMP synthase, amidophosphoribosyl-transferase (AT) and adenylosuccinate synthase were 16, 8, 107, 78 and 124 nmol/hr/mg protein, respectively, and those of the salvage enzymes, adenine, hypoxanthine and guanine phosphoribosyl-transferases, were 340, 407, and 1018 nmol/hr/mg protein, respectively. The sequence of events was elucidated after a single i.p. injection of acivicin (5 mg/kg) or tiazofurin (200 mg/kg). Within 2 hr after acivicin injection, CTP, GMP and FGAM synthases lost 85-90%, while AT and synthase II lost 50 and 80%, respectively, of their activities. The activities rose to near normal range by 72-96 hr. The bone marrow cellularity decreased, reaching a nadir at 24 and 48 hr, and returning to normal range by 72 and 92 hr; thymidine kinase activity followed a similar pattern. Tiazofurin injection depressed IMP dehydrogenase activity to 20% by 2 hr with a rebound to normal range by 48 and 72 hr. The cellularity decreased more slowly, reaching its lowest point at 24 hr and returning to normal range at 72 hr. For acivicin the marked depletion of the activities of the glutamine-utilizing enzymes and for tiazofurin that of IMP dehydrogenase might account, in part at least, for the bone marrow toxicity of these antimetabolites. Because of the presence in the bone marrow of high activities of purine and pyrimidine salvage enzymes, it should be possible to design methods utilizing nucleosides and nucleobases to protect the bone marrow from the action of antimetabolites.
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PMID:Enzymic programs of rat bone marrow and the impact of acivicin and tiazofurin. 334

Several VV structural proteins are produced by the removal of amino-terminal peptides from their cognate precursors. In the experiments reported here, directed genetic approaches were used to investigate the possible role of these terminal peptides in protein processing. As a model system, the FLAG epitope-tagged P25K precursor was used to prepare constructs in which the 31-amino-acid P25K N-terminal peptide was removed or replaced by heterologous sequences, while the -A-G*-A- cleavage motif was retained. Only a trace amount of the leaderless P25KF(delta 31) polypeptide was found within the mature virions, implying that proteolytic processing is necessary for the incorporation of the 25K product into mature virions. In trans-processing assays, significant levels of the 25K product were generated from wild-type P25KF and P4b:25KF, which consists of the 61-amino-acid P4b terminal peptide, and from P4b:25KF with 15, 30, or 44 residues of the amino terminus deleted. In contrast, only a small amount of 25K was produced from the TK:25KF, which contains the amino-terminal 30 residues of VV thymidine kinase, a protein which is not cleaved under normal circumstances. Furthermore, it has been hypothesized that a hydrophobic residue is required at position P4 relative to the -A-G*-A- motif for the cleavage to take place. An intermediate level of the 25K product was detected from the TK:25KF(Q29V) mutant which has the glutamine residue at P4 replaced with a valine residue, suggesting that the hydrophobic P4 residue and additional substrate determinants in the N-terminal peptide region are required for the proteolytic processing reaction to occur normally. Taken together, these data suggest that the amino-terminal peptides of the VV core proteins are to some extent interchangeable and that the residues proximal to the AGA site are of most importance.
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PMID:Analysis of the role of the amino-terminal peptide of vaccinia virus structural protein precursors during proteolytic processing. 787 30

(1) The currently used clinical anti-metabolites are targeted against-key enzymes of de novo purine and pyrimidine biosynthesis. However, the activities of salvage enzymes in each of the biosynthetic segments are markedly higher than those of the rate-limiting enzymes of de novo biosynthesis. Enzyme-pattern-targeted chemotherapy has been suggested to overcome the circumvention activity of salvage. Combination of inhibition of de novo and salvage pathways does provide a synergistic impact. Examples that enzyme-pattern-targeted drug treatment yields synergism include the following: tiazofurin (against IMP DH) and allopurinol (by raising serum hypoxanthine levels it inhibits GPRT); methotrexate or 5-FU lead to inhibition of the dTMP synthase reaction and AZT (a competitive inhibitor of thymidine kinase) or dipyridamole (a nucleoside transport inhibitor); acivicin, an inhibitor and inactivator of glutamine-utilizing enzymes in the de novo pathways of purine and pyrimidine biosynthesis, and dipyridamole. (2) Administration of MTX, 5-FU, tiazofurin or acivicin causes inhibition and/or inactivation of target enzymes. That these drugs are effective in spite of the presence of highly active salvage enzymes is now accounted for, at least in part, by new observations showing that these drugs markedly reduce (but do not eliminate) the activities (amounts) of CdR and TdR kinases, dTMP synthase and GPRT. This action is attributed to the rapid decay rate of these enzymes. (3) Studies on the bone marrow enzymic programs indicate that there is a window of opportunity for strengthening therapy and for the protection of bone marrow by administering salvage metabolites when the salvage enzymes are still present in high enough activities, i.e., 2-6 hr after administration of the blockers of de novo enzyme activities. (4) These results are a strong argument for discovering and utilizing inhibitors of purine and pyrimidine salvage enzymes to achieve more successful enzyme-pattern-targeted chemotherapy and to avoid development of resistant clones of cancer cells. (5) These approaches provide greater explanatory coherence than the previous accounts because recognition of (a) the importance of salvage and (b) rapid decay of key and salvage enzymes reveals a paradigm shift. The problem-solving process in chemotherapy should now be not only data-driven but also explanation-driven.
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PMID:Targeted and non-targeted actions of anti-cancer drugs. 794 86

We have identified alternatively spliced isoforms of murine Pax-3 and Pax-7 which differ by the presence or absence of a single glutamine residue in a linker region which separates two distinct DNA-binding subdomains within the paired domain. By reverse transcription-PCR, these isoforms of Pax-3 and Pax-7 (Q+ and Q-) were detected at similar levels through multiple developmental stages in the early mouse embryo. DNA-binding studies using the Q+ and Q- isoforms of Pax-3 revealed that this alternative splicing event had no major effect on the ability of these isoforms to bind to an oligonucleotide specific for the Pax-3 homeodomain (P2) or to a paired domain recognition sequence (e5) that interacts primarily with the N-terminal subdomain of the paired domain. However, DNA-binding studies with sequences (P6CON and CD19-2/A) containing consensus elements for both the N-terminal and C-terminal subdomains revealed that the Q- isoform binds to these sequences with a two- to fivefold-higher affinity; further mutation of the GTCAC core N-terminal subdomain recognition motif of CD19-2/A generated binding sites with a high degree of specificity for the Q- isoform. These differences in DNA binding in vitro were also reflected in the enhanced ability of the Q- isoform to stimulate transcription of a reporter containing multiple copies of CD19-2/A upstream of the thymidine kinase basal promoter. In support of the observations made with these naturally occurring Pax-3 isoforms, introducing a glutamine residue at the analogous position in PAX6 caused a fivefold reduction in binding to P6CON and a complete loss of binding to CD19-2/A and to the C-terminal subdomain-specific probe 5aCON. These studies therefore provide direct evidence for a role for the paired-domain linker region in DNA target site selection, and they identify novel isoforms of Pax-3 and Pax-7 that have the potential to mediate distinct functions in the developing embryo.
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PMID:An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNA-binding activity. 894 22


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