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Proceedings of the National Academy of... Dec 1975RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtilis, from both uninfected cells and...
RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) was purified from rifampicin-resistant Bacillus subtilis, from both uninfected cells and cells infected with bacteriophage SP01. The enzyme from infected cells lacked all traces of the sigma subunit, contained several polypeptides absent from the enzyme made in uninfected cells, and had an altered template specificity in a transcription assay. A cell-free protein synthesizing system from Escherichia coli, when poisoned with rifampicin, was completely dependent on addition of either of these RNA polymerase preparations for DNA-dependent protein synthesis. Under these conditions, the SP01-modified RNA polymerase preferentially stimulated the synthesis of functional mRNA for the phage enzyme dCMP deaminase (deoxycytidylate aminohydrolase, EC 3.5.4.12), whereas unmodified B. subtilis RNA polymerase could stimulate synthesis of this mRNA in small quantity and only after prolonged incubation. This mRNA belongs to a class of phage transcripts (m) which cannot be transcribed in vivo in the absence of phage-specific protein synthesis.
Topics: Aminohydrolases; Bacillus subtilis; Bacteriophages; DNA-Directed RNA Polymerases; Kinetics; Molecular Weight; Protein Biosynthesis; RNA, Messenger; Templates, Genetic; Transcription, Genetic
PubMed: 813216
DOI: 10.1073/pnas.72.12.4886 -
British Medical Journal Apr 1975Deoxycytidylate (DCMP) deaminase was assayed at various times during and after normal and abnormal pregnancies. The level in amniotic fluid was assessed at induction and... (Comparative Study)
Comparative Study
Deoxycytidylate (DCMP) deaminase was assayed at various times during and after normal and abnormal pregnancies. The level in amniotic fluid was assessed at induction and at caesarean section, and cord blood levels were estimated after normal delivery and at caesarean section. A rise occurred during labour and after hysterectomy and caesarean section--returning to normal after 2-3, and 12 days respectively. Levels above 4.8 X 10-minus 4 ml-minus 1 were found in cases of preeclamptic toxaemia and early intrauterine death and in twin pregnancies over 36 weeks' gestation. It is suggested that because of its low incidence of false-negative and false-positive results this test is far superior to other enzyme tests in pregnancy, and a further trial is in progress to assess its role.
Topics: Aminohydrolases; Amniotic Fluid; Blood; Cesarean Section; Clinical Enzyme Tests; Deoxycytidine Monophosphate; Female; Fetal Death; Humans; Hysterectomy; Labor, Induced; Labor, Obstetric; Male; Pre-Eclampsia; Pregnancy; Pregnancy Complications; Pregnancy, Multiple; Umbilical Cord
PubMed: 1137771
DOI: 10.1136/bmj.2.5961.10 -
Journal of Bacteriology Feb 1975Intact mitochondria of Neurospora crassa incorporate deoxythymidine 5'-monophosphate (dTMP) into deoxyribonucleic acid but not the label from (methyl-3H) deoxythymidine....
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.
Topics: Adenosine Triphosphate; Aminohydrolases; Cell Fractionation; Centrifugation, Density Gradient; DNA; Deoxyribonucleotides; Electron Transport Complex IV; Magnesium; Mitochondria; Neurospora; Neurospora crassa; Phosphotransferases; Potassium Chloride; Solubility; Thymidine; Thymidine Kinase; Thymidylate Synthase; Thymine Nucleotides
PubMed: 163227
DOI: 10.1128/jb.121.2.640-647.1975 -
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 -
Journal of Virology Aug 1967The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the...
The complement-fixing tumor (T) antigen induced by simian virus 40 (SV40) has been prepared from SV40-infected cell cultures, from infected cell cultures treated at the time of infection with 1-beta-d-arabinofuranosylcytosine (ara-C), and from SV40-transformed cells. Upon partial purification, the T antigen exhibited the following properties: it was tightly adsorbed by calcium phosphate gel, it was precipitated by acetic acid at pH 5 or by ammonium sulfate at about 20 to 32% saturation, and it had a molecular weight greater than 250,000, as estimated by Sephadex G-200 gel chromatography. In contrast, deoxycytidylate (dCMP) deaminase, thymidylate (dTMP) kinase, and thymidine (dT) kinase were less strongly bound to calcium phosphate and were not precipitated at pH 5; these enzymes also had much lower molecular weights than the T antigen, as did dihydrofolic (FH(2)) reductase. Furthermore, higher ammonium sulfate concentrations were required to precipitate dCMP deaminase, dTMP kinase, and FH(2) reductase activities than to precipitate the T antigen. Another difference was that the T antigen was not stabilized, but dCMP deaminase, dTMP kinase, and dT kinase, were stabilized, respectively, by dCTP, dTMP, and dT or dTTP. Deoxyribonucleic acid (DNA) polymerase activity resembled the T antigen in adsorption to calcium phosphate, in precipitation by ammonium sulfate or at pH 5, and in the rate of inactivation when incubated at 38 C. However, the polymerase activity could be partly separated from the T antigen by Sephadex G-200 gel chromatography. The cell fraction containing partially purified T antigen also contained a soluble complement-fixing antigen (presumably a subunit of the viral capsid) which reacted with hyperimmune monkey sera. The latter antigen was present in very low titers or absent from cell extracts prepared from SV40-infected monkey kidney cell cultures which had been treated with ara-C at the time of infection, or from SV40-transformed mouse kidney (mKS) or hamster tumor (H-50) cells. The T antigen, however, was present in usual amounts in SV40-transformed cells or ara-C treated, infected cells.
Topics: Aminohydrolases; Animals; Antigens; Cell Line; Cell Transformation, Neoplastic; Chromatography, Gel; Complement Fixation Tests; Cricetinae; Culture Techniques; Cytarabine; DNA Nucleotidyltransferases; Enzyme Induction; Haplorhini; Kidney; Ligases; Mice; Neoplasms; Phosphotransferases; Simian virus 40; Tetrahydrofolate Dehydrogenase; Thymidine Kinase
PubMed: 4316227
DOI: 10.1128/JVI.1.4.684-692.1967 -
Journal of Virology Feb 1967The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with...
The role of deoxyribonucleic acid (DNA) replication in the control of the synthesis of deoxycytidylate (dCMP) deaminase and lysozyme in Bacillus subtilis infected with bacteriophage 2C has been studied. These phage-induced enzymes are synthesized at different times during the latent period. It was shown by actinomycin inhibition that the formation of the late enzyme (lysozyme) required messenger ribonucleic acid (mRNA) synthesized de novo after the initiation of translation of mRNA which specifies the early function (dCMP deaminase). The inhibition of phage DNA synthesis by mitomycin C prevented the synthesis of lysozyme only when added before the onset of phage DNA replication, but it did not affect the synthesis or action of dCMP deaminase when added at any time during the latent period. Treatment of infected cells with mitomycin C after phage DNA synthesis had reached 8 to 10% of its maximal rate resulted in the production of normal amounts of lysozyme. These observations suggest that mRNA specifying early enzymes can be transcribed from parental (and probably also from progeny) DNA, whereas late functional messengers can be transcribed only after the formation of progeny DNA.
Topics: Aminohydrolases; Bacillus subtilis; Bacteriophages; Cytosine Nucleotides; DNA Replication; DNA, Viral; Dactinomycin; Mitomycins; Muramidase; RNA, Viral; Thymine; Time Factors; Tritium; Uridine
PubMed: 4990039
DOI: 10.1128/JVI.1.1.86-91.1967 -
ON THE MECHANISM OF FEEDBACK INHIBITION OF DEOXYCYTIDYLATE DEAMINASE BY DEOXYTHYMIDINE TRIPHOSPHATE.The Journal of Biological Chemistry Jul 1965
Topics: Aminohydrolases; Chemical Phenomena; Chemistry; DCMP Deaminase; Enzyme Inhibitors; Feedback; Magnesium; Nucleotides; Research; Spectrophotometry; Sulfhydryl Compounds; Thymine; Thymine Nucleotides
PubMed: 14342358
DOI: No ID Found -
The Journal of Biological Chemistry Apr 1964
Topics: Amides; Aminohydrolases; Calcium; Charcoal; Chick Embryo; Chromatography; Cobalt; Copper; Cytosine Nucleotides; DCMP Deaminase; Enzyme Inhibitors; Guanine Nucleotides; Magnesium; Manganese; Nucleotides; Pharmacology; Phosphorus Isotopes; Research; Spectrophotometry; Zinc
PubMed: 14165942
DOI: No ID Found -
STUDIES ON NORMAL AND LEUKEMIC LEUKOCYTES. VI. THYMIDYLATE SYNTHETASE AND DEOXYCYTIDYLATE DEAMINASE.The Journal of Clinical Investigation Dec 1963
Topics: Adenosine Triphosphate; Aminohydrolases; Carbon Isotopes; Chromatography; Clinical Enzyme Tests; DCMP Deaminase; Enzyme Inhibitors; Floxuridine; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukocytes; Ligases; Lymphocytes; Nucleotides; Spectrophotometry; Thymidylate Synthase
PubMed: 14086778
DOI: 10.1172/JCI104877