dCMP Deaminase Activity Research Articles

Serum deoxycytidylate deaminase as an index of high-risk pregnancy.

Serum deoxycytidylate deaminase (EC 3.5.4.12, dCMP deaminase) activity was routinely estimated over 7 years in 2460 pregnancies. The results confirm that elevated dCMP deaminase activity (greater than 4.8 units) is associated with the development of pre-eclampsia but not with essential hypertension in pregnancy; a high rising level can precede eclampsia. Elevated enzyme activity was also found in women who developed jaundice in pregnancy and in some with apparently normal pregnancies. An unexpected finding was that these so called 'false positive' high levels were associated with all the unexplained intrauterine fetal deaths that occurred later in pregnancy. Although elevated dCMP deaminase levels may be associated with a normal outcome, persisting high levels indicate the need for careful monitoring of fetal well being and, in the presence of any other sign of placental insufficiency, delivery should be expedited.

T4 ribonucleotide reductase. Physical and kinetic linkage to other enzymes of deoxyribonucleotide biosynthesis.

This laboratory has described a multienzyme aggregate from T4 phage-infected Escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and efficient delivery of DNA precursors to the replication apparatus. This paper describes improved methodology for isolation of this aggregate, and we present three lines of evidence supporting a role for ribonucleoside diphosphate reductase in functioning of the presumed complex. 1) Ribonucleoside diphosphates are readily incorporated into DNA as deoxyribonucleotides in an in situ DNA-synthesizing system from T4 phage-infected cells. 2)Ribonucleotide reductase is associated with the complex, as shown by co-sedimentation of reductase activity with other activities in the multienzyme aggregate we have described. 3)Ribonucleotide reductase is kinetically coupled to at least four other enzymes involved in a sequential pathway. The aggregated enzymes catalyze the five-step conversion of uridine diphosphate to deoxythymidine triphosphate with but a brief lag before dTTP production reaches its maximal rate. These studies have also confirmed the existence of dCTPase-dUTPase and dCMP deaminase activities in the putative complex.

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.

Deoxycytidylate deaminase activity and its template stability in human acute lymphoblastic and myeloblastic leukaemic cells

The activity of deoxycytidylate (dCMP) deaminase, an enzyme of the main pathway of the DNA synthesis and the effects of actinomycin D upon its template stability were studied in the peripheral blast cells of 11 acute leukaemia patients. The relationship between enzyme activity, template stability, cytochemical characteristics and proliferative activity of the blast cells was also investigated. By employing current cytochemical techniques, 6 of the 11 cases were classified as lymphoblastic and 5 as myeloblastic. The proliferative activity was evaluated with autoradiographic technique following short incubation with 3 H-thymidine. Acute myeloblastic leukaemia (AML) cells displayed higher enzyme activity than acute lymphoblastic leukaemia (ALL) cells. In addition, a direct correlation between the enzyme activity and the proliferative activity was shown in AML cells. dCMP deaminase template stability was assayed at the end of a 9 hr culture time in presence of actinomycin D at a dose which inhibited 90% of 3 H-uridine incorporation into the RNA of both AML and ALL cells. While none of the cases of AML showed significant change of the dCMP deaminase activity after actinomycin D treatment, in all cases of ALL a marked decrease of the enzyme activity was observed, suggesting that, at least in the steady state conditions, the AML and the ALL cells have a different rate of degradation of the RNA templates specific for the dCMP deaminase and that the rate of degradation is related to the proliferative activity of the cells. In fact, in the ALL cases a significant correlation was observed between the proliferative activity and the decrease of the enzyme activity induced by actinomycin D.

Formation of thymidine kinase and deoxycytidylate deaminase in synchronized cultures of chinese hamster cells temperature-sensitive for DNA synthesis.

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.

Analyses of deoxycytidylate deaminase molecular forms in human-mouse and monkey-mouse somatic cell hybrids.

Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosal deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey and human and monkey enzymes can be separated. To learn whether the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. these findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybird cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable.

Enzymes of deoxythymidine triphosphate biosynthesis in Neurospora crassa mitochondria

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.

Deoxycytidine Pathway in Separated Normal and Leukemic Leukocytes With Effects of Cell Culture

Abstract Enzymes of dC metabolism were studied in glass-column-separated normal and leukemic cells. Supernatants of sonicated cells were incubated with dC-3H or dCMP-3H and the end products separated with paper chromatography. Greatest recovery of dCTP, dUMP, and TTP occurred with myeloblasts and monoblasts. Mature granulocytes, and normal and leukemic lymphocytes gave the greatest breakdown to uracil. Myelocytes and lymphoblasts gave intermediate results. Leukemic lymphocytes from different patients varied greatly in their end products, but most did show evidence of TMP synthetase activity contrary to previous reports. Recovery of products of TMP synthetase and, to a lesser extent, dCMP deaminase and dC kinase activities was greatest in cells having or developing a potential for mitosis. CLL-L whose extracts showed appreciable production of dCTP, dUMP, and TdR compounds, however, did not regularly produce blast cells in response to PHA in culture. Cellular extracts of N-L, or CLL-L from cases which responded to PHA did show, on the other hand, a considerable increase in accumulation of these compounds from initial low values during culture.

Control of the synthesis of DNA precursors during Rana pipiens development

The enzymes responsible for the phosphorylation and dephosphorylation of TMP and dCMP and for the deamination of dCMP were obtained from the eggs and embryos of Rana pipiens and studied in vitro. Optimal conditions for the enzyme assays are demonstrated for both early cleaving and post-gastrula embryo homogenates. A high level of dephosphorylating activity present in post-gastrula homogenates necessitated the use of NaF in all assays of phosphorylating and deaminating activity, in order to achieve consistent results. In vitro addition of dCTP manifested a strong noncompetitive inhibition of dCMP kinase activity and, to a lesser degree, inhibition of TMP kinase while stimulating dCMP deaminase. TTP addition had no effect on dCMP or TMP kinase, but strongly inhibited dCMP deaminase activity. The absence of any demonstrable thymidine kinase activity and the availability of TMP and TTP in this system suggest that the intracellular concentration of dCTP acts as a primary regulator of DNA precursor synthesis.

Nonidentiy of some simian virus 40-induced enzymes with tumor antigen.

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.

T4 mutants unable to induce deoxycytidylate deaminase activity

T4 induces dCMP deaminase activity in Escherichia coli B. Mutants ( cd) of T4 unable to induce this activity have been isolated at a high frequency by hydroxylamine treatment. No selection procedure was needed other than an enzymatic assay of extracts of phage-infected cells. The isolation of these mutants indicates that at least one T4 cistron controls the production of dCMP deaminase activity. Studies of one cd mutant suggest that either it makes no dCMP deaminase or else one that is structurally altered. The cd + function does not appear to be essential for T4 growth in E. coli B nor does it significantly affect the production of the other early enzymes dTMP synthetase and dCMP hydrooxymethylase.