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1.
A V79 Chinese hamster fibroblast cell line selected for resistance to the toxic effects of 5-fluorouracil (Kaufman, 1984b) was found to be cross-resistant to the toxic effects of the thymidine analog 5-bromodeoxyuridine (BrdUrd). When tested for sensitivity to BrdUrd mutagenesis, the fluorouracil-resistant cells were found to be resistant to mutagenesis induced by high concentrations of BrdUrd in the medium (INC mutagenesis) but not to mutagenesis induced by the replication of DNA containing 5-bromouracil (REP mutagenesis). Analyses of deoxyribonucleoside triphosphate pools indicated that high endogenous dCTP levels in the mutant prevented the high BrdUTP/dCTP ratio associated with INC mutagenesis. However, the mutant phenotype had no effect on the nucleotide pool imbalance associated with REP mutagenesis. This mutant provides further genetic evidence for the existence of two independent mechanisms for BrdUrd mutagenesis in mammalian cells.  相似文献   

2.
A mutant murine T-cell line which overproduces purines and excretes massive quantities of inosine into the culture medium has served as a cell culture model for overproduction hyperuricemia (Ullman, B., Wormsted, M. A., Cohen, M. B., and Martin, D. W., Jr. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5127-5131). Incubation of these cells with micromolar concentrations of dipyridamole, a potent inhibitor of nucleoside transport, prevents the excretion of inosine and depresses the rate of purine biosynthesis to that of wild type cells. These concentrations of dipyridamole have no effect on cellular growth rate or on the intracellular nucleoside triphosphate or phosphoribosylpyrophosphate pools. We suggest that dipyridamole might also be useful in ameliorating purine overproduction associated with hyperuricemia and gout.  相似文献   

3.
Recent evidence proposes that the calcium-binding protein, calmodulin, plays a crucial role in the regulation or modulation of the calcium-dependent potassium conductance in Paramecium tetraurelia (Hinrichsen, R.D., Burgess-Cassler, A., Soltvedt, B.C., Hennessey, T. and Kung, C. (1986) Science 323, 503-506). We purified the calmodulins from both the wild type and pantophobiac A (a mutant lacking the above-mentioned conductance and whose phenotypic defect is traceable to its calmodulin) by hydrophobic interaction and immunoaffinity chromatographies, and examined them biochemically. In this paper we address the preliminary characterization of the two calmodulins and discuss the consequences of the genetic alteration. The differences described here are in their electrophoretic mobilities in polyacrylamide gel electrophoresis and in their binding characteristics to monoclonal antibodies raised against calmodulin from wild-type paramecia. Also, we present data which indicate a difference in the stimulation of the calmodulin-dependent enzyme bovine brain phosphodiesterase under certain conditions.  相似文献   

4.
In all previous kinetics studies of calf liver CTP synthetase, simple Michaelis-Menten hyperbolic plots were obtained. In this study it was shown that calf liver CTP synthetase could generate sigmoidal kinetic plots as a function of the substrate UTP when in the presence of the product of the reaction, CTP. The Hill number was estimated to be 2.8. The enzyme did not generate sigmoidal plots as a function of the other substrates (L-glutamine and ATP) either in the presence or absence of CTP. Thus, CTP apparently induced changes in the liver enzyme which altered the binding of UTP to the enzyme by acting at a site distinct from the UTP binding site (allosteric site). This concept was further strengthened by the fact that 3-deazaUTP, a known competitive inhibitor of the liver enzyme, did not induce sigmoidal kinetic plots. It was also shown that CTP had no effect upon the dimerization of the enzyme, thus ruling out monomer to dimer transitions as a potential mechanism for the observed sigmoidal kinetics.  相似文献   

5.
Highly purified CTP synthetase from Ehrlich ascites tumor cells catalyzes the formation of N4-substituted CTP from UTP and hydroxylamine and its derivatives. The products with hydroxylamine and O-methylhydroxylamine were identified as N4-hydroxyCTP and N4-methoxyCTP by absorption spectra and chromatographic behavior on Dowex column, respectively. The weak nucleophilic amines such as methylamine, ethylamine or diethylamine and a less nucleophilic amine, sulfamic acid, did not react with UTP. These results suggest that the nucleophilicity and basicity of amines are important in the enzymic reaction with UTP.  相似文献   

6.
Pappas A  Park TS  Carman GM 《Biochemistry》1999,38(50):16671-16677
CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] from the yeast Saccharomyces cerevisiae catalyzes the ATP-dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In this work, we demonstrated that CTP synthetase utilized dUTP as a substrate to synthesize dCTP. The dUTP-dependent activity was linear with time and with enzyme concentration. Maximum dUTP-dependent activity was dependent on MgCl(2) (4 mM) and GTP (K(a) = 14 microM) at a pH optimum of 8.0. The apparent K(m) values for dUTP, ATP, and glutamine were 0.18, 0.25, and 0.41 mM, respectively. dUTP promoted the tetramerization of CTP synthetase, and the extent of enzyme tetramerization correlated with dUTP-dependent activity. dCTP was a poor inhibitor of dUTP-dependent activity, whereas CTP was a potent inhibitor of this activity. The enzyme catalyzed the synthesis of dCTP and CTP when dUTP and UTP were used as substrates together. CTP was the major product synthesized when dUTP and UTP were present at saturating concentrations. When dUTP and UTP were present at concentrations near their K(m) values, the synthesis of dCTP increased relative to that of CTP. The synthesis of dCTP was favored over the synthesis of CTP when UTP was present at a concentration near its K(m) value and dUTP was varied from subsaturating to saturating concentrations. These data suggested that the dUTP-dependent synthesis of dCTP by CTP synthetase activity may be physiologically relevant.  相似文献   

7.
Substrate specificity of CTP synthetase from Escherichia coli   总被引:1,自引:0,他引:1  
The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.  相似文献   

8.
Sialic acids are activated by CMP-5-N-acetylneuraminic acid synthetase prior to their transfer onto oligo- or polysaccharides. Here, we present the crystal structure of the N-terminal catalytically active domain of the murine 5-N-acetylneuraminic acid synthetase in complex with the reaction product. In contrast to the previously solved structure of 5-N-acetylneuraminic acid synthetase from Neisseria meningitidis and the related CMP-KDO-synthetase of Escherichia coli, the murine enzyme is a tetramer, which was observed with the active sites closed. In this conformation a loop is shifted by 6A towards the active site and thus an essential arginine residue can participate in catalysis. Furthermore, a network of intermolecular salt-bridges and hydrogen bonds in the dimer as well as hydrophobic interfaces between two dimers indicate a cooperative behaviour of the enzyme. In addition, a complex regulation of the enzyme activity is proposed that includes phosphorylation and dephosphorylation.  相似文献   

9.
10.
A mutation in the structural gene coding for seryl-tRNA synthetase in temperature-sensitive Escherichia coli K28 has been reported to alter the level of enzyme expression at high temperature (R. J. Hill and W. Konigsberg, J. Bacteriol. 141:1163-1169, 1980). We identified this mutation as a C-->T transition in the first base of codon 386, resulting in a replacement of histidine by tyrosine. The steady-state levels of serS mRNA in K28 and in the wild-type strains are very similar. Pulse-chase labeling experiments show a difference in protein stability, but not one important enough to account for the temperature sensitivity of K28. The main reason for the temperature sensitivity of K28 appears to be the low level of specific activity of the mutant synthetase at nonpermissive temperature, not a decreased expression level. Spontaneous temperature-resistant revertants were selected which were found to have about a fivefold-higher level of SerRS than the K28 strain. We identified the mutation responsible for the reversion as being upstream from the -10 sequence in the promoter region. The steady-state levels of serS mRNA in the revertants are significantly higher than that in the parental strain.  相似文献   

11.
Cyclopentenylcytosine (CPEC) is phosphorylated in L1210 cells with CPEC triphosphate as the major metabolite. Partially purified uridine-cytidine kinase catalyzes the initial phosphorylation of cyclopentenylcytosine with an apparent Km of 196 +/- 9 microM, and cyclopentenylcytosine is a competitive inhibitor of cytidine phosphorylation by this enzyme with a Ki value of 144 +/- 14 microM. Examination of the CTP synthetase activity in extracts of L1210 cells revealed a dose-dependent decrease on exposure of cells to CPEC. Synthesis of CPEC triphosphate by an enzymatic method permitted direct examination of the inhibition of partially purified CTP synthetase. CPEC triphosphate inhibited bovine CTP synthetase with a median inhibitory concentration of 6 microM, whereas CPEC mono- and diphosphates were ineffective. CTP synthetase showed a classical Michaelis-Menten hyperbolic plot of velocity and UTP concentration in the presence of saturating concentrations of ATP and glutamine, but CPEC triphosphate induced sigmoidal kinetic plots. The Hill coefficient was calculated to be 3.2.  相似文献   

12.
Nucleotide sequence of Escherichia coli pyrG encoding CTP synthetase   总被引:18,自引:0,他引:18  
The amino acid sequence of Escherichia coli CTP synthetase was derived from the nucleotide sequence of pyrG. The derived amino acid sequence, confirmed at the N terminus by protein sequencing, predicts a subunit of 544 amino acids having a calculated Mr of 60,300 after removal of the initiator methionine. A glutamine amide transfer domain was identified which extends from approximately amino acid residue 300 to the C terminus of the molecule. The CTP synthetase glutamine amide transfer domain contains three conserved regions similar to those in GMP synthetase, anthranilate synthase, p-aminobenzoate synthase, and carbamoyl-P synthetase. The CTP synthetase structure supports a model for gene fusion of a trpG-related glutamine amide transfer domain to a primitive NH3-dependent CTP synthetase. The major 5' end of pyrG mRNA was localized to a position approximately 48 base pairs upstream of the translation initiation codon. Translation of the gene eno, encoding enolase, is initiated 89 base pairs downstream of pyrG. The pyrG-eno junction is characterized by multiple mRNA species which are ascribed to monocistronic pyrG and/or eno mRNAs and a pyrG eno polycistronic mRNA.  相似文献   

13.
14.
Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min(-1) and a K(I) of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with beta-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The beta-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its beta-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the beta-elimination product of oCTP.  相似文献   

15.
Alkaline phosphatase is one of several enzymes that accumulate in a temporally regulated sequence during the development of Dictyostelium discoideum. These enzymes can be used to monitor specific gene expression; moreover, isolation and analysis of mutations in the structural gene(s) can serve to indicate some of the essential steps in programmed synthesis and morphogenesis. A mutation (alpA) which affects the activity and substrate affinity of alkaline phosphatase was isolated in D discoideum using a procedure for screening large numbers of clones. Alkaline phosphatase activity at all stages of vegetative growth and development was altered by the mutation. Several physical properties of the enzyme from growing cells and developed cells were compared and found to be indistinguishable. It is likely that a single enzyme is responsible for the majority of alkaline phosphatase activity in growth and development. The mutation is coexpressed in diploids heterozygous for alpA and maps to linkage group III. One of the haploid segregants isolated from these diploids carries convenient markers on each of the six defined linkage groups and can be used for linkage analysis of other genetic loci.  相似文献   

16.
The development and marketing of ‘novel’ genetically modified (GM) crops in which composition has been deliberately altered poses a challenge to the European Union (EU)'s risk assessment processes, which are based on the concept of substantial equivalence with a non‐GM comparator. This article gives some examples of these novel GM crops and summarizes the conclusions of a report that was commissioned by the European Food Safety Authority on how the EU's risk assessment processes could be adapted to enable their safety to be assessed.  相似文献   

17.
From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.  相似文献   

18.
The regulatory role of the allosteric site of CTP synthetase on flux through the enzyme in situ and on pyrimidine nucleotide triphosphate (NTP) pool balance was investigated using a mutant mouse T lymphoblast (S49) cell line which contains a CTP synthetase refractory to complete inhibition by CTP. Measurements of [3H]uridine incorporation into cellular pyrimidine NTP pools as a function of time indicated that CTP synthesis in intact wild type cells was markedly inhibited in a cooperative fashion by small increases in CTP pools, whereas flux across the enzyme in mutant cells was much less affected by changes in CTP levels. The cooperativity of the allosteric inhibition of the enzyme was greater in situ than in vitro. Exogenous manipulation of levels of GTP, an activator of the enzyme, indicated that GTP had a moderate effect on enzyme activity in situ, and changes in pools of ATP, a substrate of the enzyme, had small effects on CTP synthetase activity. The consequences of incubation with actinomycin D, cycloheximide, dibutyryl cyclic AMP, and 6-azauridine on the flux across CTP synthetase and on NTP pools differed considerably between wild type and mutant cells. Under conditions of growth arrest, an intact binding site for CTP on CTP synthetase was required to maintain a balance between the CTP and UTP pools in wild type cells. Moreover, wild type cells failed to incorporate H14CO3- into pyrimidine pools following growth arrest. In contrast, mutant cells incorporated the radiolabel at a high rate indicating loss of a regulatory function. These results indicated that uridine nucleotides are important regulators of pyrimidine nucleotide synthesis in mouse S49 cells, and CTP regulates the balance between UTP and CTP pools.  相似文献   

19.
In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.  相似文献   

20.
5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K(198)RXR to be essential for nuclear transport and Arg(202) to be necessary to complete the transport process. Cytoplasmic forms of CMP-Neu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg(199) and Arg(202) are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMP-sialic acid synthetases reveals Arg(202) and Gln(203) as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.  相似文献   

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