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The Biochemical Journal Nov 1974The activities of dCMP deaminase and DNA polymerase I increased twofold and fivefold in BHK-21/C13 cells after infection by the virus of herpes simplex. The increases...
The activities of dCMP deaminase and DNA polymerase I increased twofold and fivefold in BHK-21/C13 cells after infection by the virus of herpes simplex. The increases were greatly diminished, and under certain conditions prevented, by inclusion of actinomycin D or cycloheximide in the cell-virus system during the infective cycle. The dCMP deaminase purified from infected cells harvested 8h after infection differed from the deaminase purified from non-infected cells inasmuch as (a) it was more resistant to heating at 37 degrees C; (b) the substrate (dCMP) concentration at half-maximum velocity was lower; (c) maximum activation was achieved by a lower concentration of dCTP; (d) it was more resistant to inhibition by dTTP; and (e) it behaved differently when assayed in the presence of a herpes-virus-specific antiserum. The DNA polymerase activity in the infected cells was markedly decreased in the presence of the herpes-virus-specific antiserum.
Topics: Aminohydrolases; Animals; Cell Line; Cells, Cultured; Cricetinae; Cycloheximide; Cytosine Nucleotides; DNA Nucleotidyltransferases; Dactinomycin; Deoxycytidine Monophosphate; Enzyme Induction; Hot Temperature; Immune Sera; Kidney; Kinetics; Simplexvirus
PubMed: 4376945
DOI: 10.1042/bj1430403 -
The Biochemical Journal Jul 1974dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient...
dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40mum, gave a hyperbolic curve for activity versus dCMP concentration and a K(m) value for dCMP of 0.91mm. dCTP activation required the presence of Mg(2+) or Mn(2+) ions. dTTP inhibited the deaminase maximally at 15mum; the inhibition required the presence of Mg(2+) or Mn(2+) ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).
Topics: Aminohydrolases; Animals; Cell Line; Centrifugation, Density Gradient; Chromatography, DEAE-Cellulose; Chromatography, Gel; Cricetinae; Culture Techniques; Cytosine Nucleotides; Deoxyribonucleotides; Ethylenes; Glycols; Hydrogen-Ion Concentration; Kidney; Kinetics; Magnesium; Manganese; Molecular Weight; Temperature; Thymine Nucleotides
PubMed: 4455201
DOI: 10.1042/bj1410211 -
FEBS Letters May 2002Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation...
Human mature sperm cells have a high nuclease and 5-methyldeoxycytidine monophosphate (5-mdCMP) deaminase activity. The deaminase converts the nuclease degradation product 5-mdCMP into dTMP which is further cleaved into thymine and the abasic sugar-phosphate. Both 5-methylcytidine 5' and 3' monophosphates are good substrates for the deaminase. 5-methylcytidine is not a good deaminase substrate and 5-methylcytosine (5mC) is not a substrate. A purified fraction of the deaminase free of nucleases deaminates 5mC present in intact methylated double-stranded DNA. 5-mdCMP deaminase co-purifies on SDS-PAGE with dCMP deaminase and has an apparent molecular weight of 25 kDa. The enzyme requires no divalent cations and has a Km of 1.4 x 10(-7) M for 5-mdCMP and a Vmax of 7 x 10(-11) mol/h/microg protein. The possible biological implications of the deaminase's activities in the present system are discussed.
Topics: Aminohydrolases; Cytidine; DNA; Deoxycytidine Monophosphate; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Humans; Male; Molecular Weight; Oligonucleotides; Spermatozoa; Substrate Specificity; Thymine; Uracil
PubMed: 12023031
DOI: 10.1016/s0014-5793(02)02737-0 -
Biochemical Pharmacology Mar 1980Cytidine deaminase, an enzyme found in the supernatant fluid of hepatocytes, granulocytes and tumor cells, and in plasma, degrades the antitumor agents cytosine...
Cytidine deaminase, an enzyme found in the supernatant fluid of hepatocytes, granulocytes and tumor cells, and in plasma, degrades the antitumor agents cytosine arabinoside and 5-azacytidine. Uridine and its analogs, 3-deazauridine, 5-bromodeoxyuridine, 5-fluorodeoxyuridine and 6-azauridine, were found to competitively inhibit cytidine deaminase; the most potent inhibitor was 3-deazauridine (K(i) = 1.9 x 10(-5) M). In addition, deoxycytidylate deaminase, which degrades cytosine arabinoside monophosphate to the inactive uracil arabinoside monophosphate (K(m) = 9 x 10(-4) M), was competitively inhibited by 3-deazauridine monophosphate, as well as by the nucleotides of other uridine analogs. These results suggest that uridine analogs such as 3-deazauridine may have value in protecting cytosine arabinoside, 5-azacytidine and their monophosphate nucleotides from degration by neucleoside and nucleotide deaminases.
Topics: 3-Deazauridine; Azauridine; Bromodeoxyuridine; Cytidine Deaminase; DCMP Deaminase; Floxuridine; Humans; Kinetics; Uridine
PubMed: 20227960
DOI: 10.1016/0006-2952(80)90561-4 -
British Journal of Cancer Apr 2018Deoxycytidylate deaminase (DCTD) and ribonucleotide reductase subunit M1 (RRM1) are potential prognostic and predictive biomarkers for pyrimidine-based chemotherapy in... (Observational Study)
Observational Study
Intratumoural expression of deoxycytidylate deaminase or ribonuceotide reductase subunit M1 expression are not related to survival in patients with resected pancreatic cancer given adjuvant chemotherapy.
BACKGROUND
Deoxycytidylate deaminase (DCTD) and ribonucleotide reductase subunit M1 (RRM1) are potential prognostic and predictive biomarkers for pyrimidine-based chemotherapy in pancreatic adenocarcinoma.
METHODS
Immunohistochemical staining of DCTD and RRM1 was performed on tissue microarrays representing tumour samples from 303 patients in European Study Group for Pancreatic Cancer (ESPAC)-randomised adjuvant trials following pancreatic resection, 272 of whom had received gemcitabine or 5-fluorouracil with folinic acid in ESPAC-3(v2), and 31 patients from the combined ESPAC-3(v1) and ESPAC-1 post-operative pure observational groups.
RESULTS
Neither log-rank testing on dichotomised strata or Cox proportional hazard regression showed any relationship of DCTD or RRM1 expression levels to survival overall or by treatment group.
CONCLUSIONS
Expression of either DCTD or RRM1 was not prognostic or predictive in patients with pancreatic adenocarcinoma who had had post-operative chemotherapy with either gemcitabine or 5-fluorouracil with folinic acid.
Topics: Adenocarcinoma; Adult; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Chemotherapy, Adjuvant; DCMP Deaminase; Disease-Free Survival; Humans; Immunohistochemistry; Pancreatectomy; Pancreatic Neoplasms; Prognosis; Randomized Controlled Trials as Topic; Ribonucleoside Diphosphate Reductase; Tissue Array Analysis; Tumor Suppressor Proteins
PubMed: 29523831
DOI: 10.1038/s41416-018-0005-1 -
Open Biology Jan 20165-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a...
5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.
Topics: Antibodies; Bromodeoxyuridine; Cell Death; Cell Line, Tumor; Cell Nucleus; Cytidine Deaminase; DNA; DNA Replication; Deoxycytidine; Deoxyuridine; Humans; Metabolome; RNA, Small Interfering
PubMed: 26740587
DOI: 10.1098/rsob.150172 -
Cancer Research Feb 19922',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition...
2',2'-Difluorodeoxycytidine (dFdC, Gemcitabine) is a deoxycytidine analogue which, after phosphorylation to the 5'-di- and 5'-triphosphate (dFdCTP), induces inhibition of DNA synthesis and cell death. We examined the values for elimination kinetics of cellular dFdCTP and found they were dependent on cellular concentration after incubation of CCRF-CEM cells with dFdC and washing into drug-free medium. When the drug was washed out at low cellular dFdCTP levels (less than 50 microM), dFdCTP elimination was linear (t1/2 = 3.3 h), but it became biphasic at intracellular dFdCTP levels greater than 100 microM. Although the initial elimination rate was similar at all concentrations, at higher concentrations the terminal elimination rate increased with increasing cellular dFdCTP concentration, with a nearly complete inhibition of dFdCTP elimination at 300 microM. The deamination product 2',2'-difluorodeoxyuridine was the predominant extracellular catabolite at low cellular dFdCTP concentrations, whereas at high dFdCTP concentrations dFdC was the major excretion product. The dCMP deaminase inhibitor 3,4,5,6-tetrahydrodeoxyuridine transformed the monophasic dFdCTP degradation seen at low dFdCTP levels into a biphasic process, whereas the deoxycytidine deaminase inhibitor 3,4,5,6-tetrahydrouridine had no effect on dFdCTP elimination. An in situ assay indicated that dCMP deaminase activity was inhibited in whole cells, an action that was associated with a decreased dCTP:dTTP value. In addition, dFdCTP inhibited partially purified dCMP deaminase with a 50% inhibitory concentration of 0.46 mM. We conclude that dFdC-induced inhibition of dCMP deaminase resulted in a decrease of dFdCTP catabolism, contributing to the concentration-dependent elimination kinetics. This action constitutes a self-potentiation of dFdC activity.
Topics: Antimetabolites, Antineoplastic; Cell Line; Cytidine Triphosphate; DCMP Deaminase; Deoxycytidine; Deoxyribonucleotides; Humans; Kinetics; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Ribonucleotides; Gemcitabine
PubMed: 1732039
DOI: No ID Found -
The Journal of Biological Chemistry Jan 2015Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here...
Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.
Topics: Allosteric Regulation; Allosteric Site; Amino Acid Sequence; Bacteriophages; Crystallography, X-Ray; DCMP Deaminase; Deoxycytosine Nucleotides; Enzyme Activation; Enzyme Inhibitors; Escherichia coli; Gene Expression; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Recombinant Proteins; Sequence Alignment; Substrate Specificity; Thymine Nucleotides; Tyrosine; Viral Proteins
PubMed: 25404739
DOI: 10.1074/jbc.M114.617720 -
Molecular and Cellular Biochemistry Jul 1987Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic...
Deoxycytidylate deaminase isolated from normal human lymphocytes and from mononuclear leucocytes from patients with acute lymphoblastic leukemia, chronic lymphocytic leukemia and acute monocytic leukemia has been characterized in regard to the substrate, dAMP and the allosteric regulators dCTP and dTTP. The enzymes exhibited sigmoidal initial velocity versus dCMP concentration whereas in the presence of the activator, dCTP, Michaelis-Menten kinetics were obtained. At saturating substrate concentrations dTTP acted as an allosteric inhibitor of the enzyme isolated from non-stimulated as well as from stimulated lymphocytes. However, the enzymes isolated from the leukemic cells had lost the allosteric regulation by dTTP. At low substrate concentrations the competitive inhibitor, dAMP, activated all the enzymes. This activation was abolished in the presence of dCTP which indicates that dAMP might be involved in the regulation of dCMP deaminase activity and thus influence the dCTP and dTTP pools under physiological conditions.
Topics: Aged; DCMP Deaminase; Deoxyribonucleotides; Female; Humans; In Vitro Techniques; Leukemia; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Nucleotide Deaminases; Phytohemagglutinins
PubMed: 3627112
DOI: 10.1007/BF00219395 -
Biochimica Et Biophysica Acta Mar 1993Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column,... (Comparative Study)
Comparative Study
Deoxycytidylate deaminase was purified about 7000-fold to homogeneity from a human source (HeLa cells). The final step in the purification employed an affinity column, which increased the specific activity of the enzyme from the previous step by 500-fold. Similar to most other dCMP deaminases, this enzyme is allosterically regulated by microM levels of dCTP and dTTP. However, unlike the other enzymes the most dramatic allosteric responses occur at substrate levels of 0.1 mM dCMP or less, where at least a 10-fold increase in activity is effected by dCTP. The enzyme is particularly sensitive to inhibition by dTTP with 50% inhibition being obtained at 1.5 x (10(-6) M in the absence of dCTP. Antibody to the human enzyme did not cross-react with a dCMP deaminase induced in Escherichia coli by T4-bacteriophage, nor did antibody to the phage-induced enzyme cross-react with the human deaminase. A potential transition-state analogue of the substrate, 2'-beta-D-deoxyribose-pyrimidin-2-one 5'-phosphate was prepared, and found to inhibit dCMP deaminase competitively with a Ki of 1.2 x 10(-8) M.
Topics: Antibodies; Antibody Specificity; Chromatography, Affinity; Cross Reactions; DCMP Deaminase; Deoxycytidine Monophosphate; Escherichia coli; HeLa Cells; Humans; Hydrogen-Ion Concentration; Substrate Specificity; Thymine Nucleotides
PubMed: 8448179
DOI: 10.1016/0167-4838(93)90143-f