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:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine. Mitochondrial homogenates contain deoxythymidylate kinase (EC 2.7.4.9), deoxycytidylate aminohydrolase (dCMP deaminase) (EC 3.5.4.12), and thymidylate synthetase (EC 2.1.1b), but not thymidine kinase (EC 2.7.1.21) activity. dTMP kinase is loosely bound to the mitochondrial membrane and is solubilized by 0.4 M KCl in mitochondrial homogenates, the dCMP aminohydrolase deaminase) is bound to the inner membrane and is not solubilized by 0.4 M KCl. dTMP synthetase activity is found in the 2,000 times g particulate fractions by homogenization of mitochondria in 0.4 M KCl. The dCMP deaminase activity found in the particulate fraction of the inner membrane is efficiently regulated by the products of the pathway: deoxycytidine 5'-triphosphate activates whereas deoxythymidine 5'-triphosphate inhibits, as found for the soluble enzyme from other sources. These data indicate that mitochondria of N. crassa contain specific enzymes for the biosynthesis of deoxythymidine triphosphate.
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PMID:Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria. 16 27

Cytosol thymidine kinase (TK) and deoxycytidylate (dCMP) deaminase formation was investigated in synchronized cultures of K12 Chinese hamster cells which have a temperature-sensitive lesion affecting the initiation of DNA synthesis. Enzyme formation was found to be cycloheximide-sensitive and also temperature-dependent. Beginning at about six hours after addition of medium with 10% calf serum to serum-depleted K12 cultures, cytosol TK and dCMP deaminase activities increased when the cultures were incubated at 36.5 degrees but not at 40.5 degrees. When cultures were shifted from 36.5 degrees to 40.5 degrees at 4,6, or 8 hours after serum addition, TK activity continued to increase, though not to the level observed at ten hours in cultures maintained at 36.5 degrees. Actinomycin D addition at the time of serum reversal or four hours later blocked the TK increase normally observed at the permissive temperature at ten hours. However, when actinomycin D addition was delayed for six or eight hours after serum addition, the increase in TK measured at ten hours resembled that observed in the temperature shift-up experiments. The results provide evidence that the mutation in K12 Chinese hamster cells most likely blocks the progression through G1 into S and suggest that transcription or post-transcriptional processing required for TK formation is affected.
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PMID:Formation of thymidine kinase and deoxycytidylate deaminase in synchronized cultures of chinese hamster cells temperature-sensitive for DNA synthesis. 126 6

5-Ethynyl-1-beta-D-arabinofuranosylcytosine (EAC) was prepared from 1-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)cytosine by iodination followed by coupling with (trimethylsilyl)acetylene and deblocking. At 50 microM, EAC was found to inhibit the in vitro replication of herpes simplex virus type 1 and type 2 by greater than 99%. EAC also showed activity against a strain of HSV-1 resistant to (E)-5-(2-bromovinyl)-2'-deoxyuridine which has an alteration of the virus-induced thymidine kinase (TK). At 100 microM, EAC did not inhibit the in vitro growth of leukemia L1210 and HeLa cells. EAC was resistant to the action of dCR-CR deaminase, its rate of deamination being approximately 2% that of dCR. The compound was a poor substrate for dCR kinase, but it was phosphorylated by HSV-1- and HSV-2-induced TKs at 50% and 30%, respectively, the rate of thymidine.
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PMID:Acetylenic nucleosides. 4. 1-beta-D-arabinofuranosyl-5-ethynylcytosine. Improved synthesis and evaluation of biochemical and antiviral properties. 282 32

Thymidine kinase was induced after infection of an established strain of green monkey kidney cells (CV-1) with simian adenovirus SV15. Increased levels of thymidine kinase were first observed 8 to 10 hr postinoculation (PI), and the levels increased four- to eightfold by 16 to 24 hr PI. A transient increase (1.5- to 3-fold) of deoxyribonucleic acid (DNA) polymerase activity was also observed about 18 hr PI, but the level of deoxycytidylic deaminase was not enhanced. The inductions of thymidine kinase and DNA polymerase were not obtained when protein synthesis was inhibited with 10(-5) M cycloheximide. However, the enzyme increases did take place when infected cultures were treated with 1-beta-D-arabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis and SV15 replication. The incorporation of tritium-labeled thymidine (H(3)-dT) into DNA was also stimulated 8 to 24 hr after infection with SV15.
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PMID:Enzyme induction in green monkey kidney cultures infected with simian adenovirus. 562 53

Deoxyribonucleic acid (DNA) polymerase activity was induced at approximately 18 to 20 hr after infection of secondary cultures of human embryonic kidney cells with adenovirus type 2 or type 12, and, at 30 to 50 hr after infection, the activity of this enzyme increased two- to threefold. The activity of thymidine kinase was also induced, but the activity of deoxycytidylic deaminase was not. The DNA content per cell at 71 hr after infection was 1.6-fold greater in adenovirus 2-infected cultures, and approximately 2.4-fold greater in adenovirus 12-infected cultures, than in the noninfected cultures. Several properties of DNA polymerase were studied. The enzymes in normal and adenovirus 2- or 12-infected cell extracts were saturated by approximately the same concentration of heat-denatured salmon sperm DNA primer (160 mug/ml); the enzyme activities had a similar broad pH optimum between 7.5 and 9. Extracts prepared from cells infected by either adenovirus did not activate DNA polymerase from noninfected cells, nor did the noninfected cell extracts inhibit enzyme activity of infected cell extracts. DNA polymerase in both normal and adenovirus 2- or 12-infected cells was located predominantly in the nucleus. In each case, the cytoplasm had only 30% of the enzyme activity of the nucleus. At 40 hr after infection with adenovirus 2 or 12, the activities of the enzyme in the nuclear and cytoplasmic fractions increased two- to threefold. Puromycin, an inhibitor of protein synthesis, prevented DNA polymerase induction when added to cultures during the 18- to 30-hr postinfection period, and it arrested the additional increase in enzyme activity when added after enzyme induction began. However, the increases in both DNA polymerase and thymidine kinase activities took place after treatment of infected cultures with 1-beta-d-arabinofuranosylcytosine, an inhibitor of DNA synthesis and adenovirus growth.
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PMID:Enhanced deoxyribonucleic acid polymerase activity in human embryonic kidney cultures infected with adenovirus 2 or 12. 574 41

Deoxycytidine (dC) deaminase activity has been previously reported to be induced in herpes simplex virus (HSV)-infected cells (Chan, 1977). In contrast, we report here that HSV infection of either hamster cells naturally deficient in this enzyme activity or mouse cells containing a low level of activity never resulted in appearance of stimulation of dC deaminase, whereas thymidine kinase (TK) was always induced. Surprisingly, dC deaminase activity, which differed by electrophoretic mobility from the mouse or human cell enzyme, was discovered in some cells selected for the presence of HSV TK after infection with u.v.-irradiated HSV. Evidence is presented which suggests that the appearance of this new enzyme was not due to the presence of virus genes but rather to mycoplasma contamination.
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PMID:Analysis of deoxycytidine (dC) deaminase activity in herpes simplex virus-infected or HSV TK-transformed cells: association with mycoplasma contamination but not with virus infection. 627 19

The incorporation into DNA of 5-bromocytosine and 5-iodocytosine, derived from their respective administered deoxyribonucleoside analogs, has been demonstrated in studies with cells infected with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and in cells transformed with the thymidine kinase gene of HSV-1. No significant incorporation of iodocytosine or iodouracil occurred in the DNA of uninfected or nontransformed cells when the deaminating enzymes were inhibited, in accord with past studies in our laboratory with 5-bromodeoxycytidine and tetrahydrouridine. When 2'-deoxytetrahydrouridine, a potent inhibitor of cytidine deaminase and dCMP deaminase, was utilized, all the counts in DNA that were derived from [(125)I]iododeoxycytidine appeared as iodocytosine in HSV-infected cells. In the absence of a deaminase inhibitor, 32 to 45% of the counts associated with DNA pyrimidines appeared as iodocytosine, and 55 to 68% appeared as iodouracil in HSV-infected cells. Substantial incorporation of iodocytosine (16%) occurred in cells transformed with the HSV thymidine kinase gene, suggesting the importance of the specificity of cellular nucleoside kinases and the activity of the deaminases in presenting unmodified bases to an undiscriminating polymerase. Incorporation into DNA of bromocytosine derived from [(3)H]bromodeoxycytidine was demonstrated in HSV-2 infected cells; very little incorporation of bromocytosine compared with bromouracil could be demonstrated in these cells in the absence of inhibition of the deaminases (19% of the total counts associated with pyrimidines with deaminase inhibition and 1.5% without). Limited studies with 5-methyl[5-(3)H]deoxycytidine indicated essentially no (or very little) incorporation of this analog as such in the DNA of HSV-1- and HSV-2-infected and -transformed cells. This suggests an exclusion or repair mechanism preventing inappropriate methylcytosine incorporation in DNA. The addition of nucleoside and deoxyribonucleoside deaminase inhibitors, which leads to the incorporation of 5-halogenated analogs of deoxycytidine into DNA as such, does not impair their antiviral activity. We infer from studies with 4-N-alkyl (ethyl and isopropyl)-substituted analogs of iododeoxycytidine that they are incorporated as such into DNA without deamination and effectively inhibit the virus at concentrations that are marginally toxic. Among the several reasons presented for the heightened potential efficacy of analogs of deoxycytidine compared with those of deoxyuridine is that the former, as analogs of 5-methyldeoxycytidine, may impair viral replication by perturbing processes involving methylation and changes in the methylation of deoxycytidine in DNA which appear to be important for the process of HSV maturation. In addition, this capacity to perturb methylation may, in turn, be the key to their potential as agents affecting entry into or emergence from latency, a process in which dramatic changes in the postpolymer 5-methylation of deoxycytidine occur in the DNA of herpesviruses.
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PMID:Incorporation of 5-substituted analogs of deoxycytidine into DNA of herpes simplex virus-infected or - transformed cells without deamination to the thymidine analog. 630 14

To ascertain whether concomitant expression of Escherichia coli deaminase (CD) and herpes simplex virus type-1 thymidine kinase (HSV-1 TK) could mediate greater levels of cytotoxicity beyond that observed with either suicide gene alone, 9L gliosarcoma cells were transduced with a retrovirus encoding a CD/HSV-1 TK fusion gene. The resultant CD/HSV-1 TK fusion protein (CDglyTK) was found to be bifunctional via CD and HSV-1 TK enzymatic assays, and conferred upon cells prodrug sensitivities equivalent to or better than that observed for each enzyme independently (ganciclovir [GCV] and bromovinyldeoxyuridine [BVdU] for HSV-1 TK and 5-fluorocytosine [5-FC] for CD). Simultaneous treatment of CDglyTK-expressing cells with prodrugs specific for HSV-1 TK and CD (GCV/5-FC or BVdU/5-FC) resulted in slight synergistic toxicity, two- to three-fold greater than that expected if the cytotoxic effects of each prodrug were purely additive. More importantly, co-treatment with HSV-1 TK- and CD-specific prodrugs was found to increase greatly the radiosensitivity of CDglyTK-expressing cells. Sensitivity enhancement ratios of 2.44 (GCV/5-FC) and 3.90 (BVdU/5-FC) were achieved. The results suggest that double suicide gene therapy, using a bifunctional CD/HSV-1 TK fusion gene, coupled with radiotherapy may provide a highly efficient means of selectively treating cancer.
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PMID:Glioma cells transduced with an Escherichia coli CD/HSV-1 TK fusion gene exhibit enhanced metabolic suicide and radiosensitivity. 898 97

Recently, it has been shown, that 2-Chloro-deoxyadenosine (1), a series of analogues, and other DNA synthesis inhibitors, increased the deoxycytidine kinase (dCK) enzyme activity in different cells, without influencing thymidine kinase isoenzymes (TK1, TK2), dCMP-deaminase and thymidylate synthase (TS) activities (2,3). The dCK activity was 2-4 times higher in analogue treated cells, than in controls, which can not be explained by metabolic pool imbalance induced by the drugs. New mRNA and protein synthesis of dCK could not be detected, thus post-translational modification has been suggested for potentiation the activity of the dCK (1). Because secondary modifications of enzymes usually involve the signalling processes in cells, the universal G-protein activator fluorine ions were tested. dCK activity of human lymph node lymphocytes were increased 2-times, if cells were incubated in the presence of NaF for 1-2 hrs in cultures, while TK activity was not changed. The formation of dUTP from dCyd, was also enhanced by NaF, in parallel of dCK potentiation.
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PMID:Deoxycytidine kinase can be also potentiated by the G-protein activator NaF in cells. 959 3

DNA mismatch repair (MMR) defects bring about a strong mutator phenotype and microsatellite instability (MSI). In an attempt to exploit MSI in cancer therapy, we constructed expression vectors carrying a thymidine kinase/blasticidin deaminase fusion gene downstream from a (C)(12) or an (A)(26) microsatellite and stably transfected these constructs into human cells in which the MMR status could be regulated by doxycycline. We now show that ganciclovir-resistant clones arising through frameshifts in the (C)(12) microsatellite were 20 times more frequent in cells in which MMR was inactivated. This difference may be exploited in gene therapy of tumors with MSI, which represent a substantial proportion of cancers of many different tissues.
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PMID:Differential killing of mismatch repair-deficient and -proficient cells: towards the therapy of tumors with microsatellite instability. 1467 62


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