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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

2 theta-C-methyl substituted and phosphonate analogs of UTP were prepared and together with the synthesized earlier 3'-C-methyl-UTP were investigated in the RNA synthesis reaction catalysed by Escherichia coli RNA-polymerase. Substrate properties of UTP analogs were studied in the presence of all natural triphosphates, in the absence of UTP and under conditions of soil substrate reaction. It was shown that UTP(3'CH3) is incorporated into the RNA chain and terminates further RNA elongation. Another analog UTP (2'CH3) may substitute natural UTP in RNA synthesis, but the effectivity of its incorporation is diminished. Phosphonate analog UTP(5'CH2) is a pseudoterminator of RNA synthesis. The conformational analysis of 2'- and 3'C-methylnucleosides by force-field method of calculation was carried out in order to find energetically forbidden conformations of these analogs due to the collision of bulky methyl group and a heterocyclic base. An attempt was made to fix the conformation of the substrate during its enzymatic transformation.
Mol Biol (Mosk)
PMID:[Substrate properties of C'-methylnucleoside triphosphates in a reaction of RNA synthesis catalyzed by Escherichia coli RNA-polymerase]. 248 43

The allosteric control of aspartate transcarbamoylase (ATCase, EC 2.1.3.2) of Escherichia coli involves feedback inhibition by both CTP and UTP rather than just CTP alone. It has been known that CTP functions as a heterotropic inhibitor of catalysis; however, the inhibition by CTP alone is incomplete (50-70% at various aspartate concentrations) and there is only a partial occupancy of the allosteric binding sites by CTP at saturating concentrations. The logic of these allosteric characteristics can now be understood in that UTP is a synergistic inhibitor of ATCase in the presence of CTP even though UTP has no independent effect at pH 7.0. When saturating concentrations of CTP are present, the concentration of substrate required for half-maximal activity (S0.5) of the native holoenzyme for aspartate increases from 5 to 11 mM. When CTP and UTP are both present, the aspartate requirement increases further (S0.5 = 17 mM). At aspartate concentrations less than 5 mM, the heterotropic inhibition of ATCase is 90-95% in the presence of both pyrimidine nucleotides. UTP does enhance the binding of CTP to the holoenzyme but the number of tight binding sites does not change (n = 3). The binding of UTP is stabilized in the presence of CTP although its binding characteristics are not as strong as those of CTP. The recent crystallographic studies of Kim et al. [Kim, H.K., Pan, Z., Honzatko, R.B., Ke, H.M. & Lipscomb, W.N. (1987) J. Mol. Biol. 196, 853-875] have described a structural asymmetry across the molecular two-fold axis that is consistent with these CTP/UTP interactions. The synergistic inhibition of ATCase by both CTP and UTP provides a satisfying logic for ensuring a balance of endogenous pyrimidine nucleotide pools.
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PMID:In the presence of CTP, UTP becomes an allosteric inhibitor of aspartate transcarbamoylase. 264 6

The URA5 gene of Saccharomyces cerevisiae encodes orotate phosphoribosyl transferase (EC 2.4.2.10; OPRTase) which catalyses the transformation of orotate to OMP in the pyrimidine pathway. We present in this paper the cloning and the sequencing of this gene, the last in the yeast pyrimidine pathway to be cloned. We have deduced the protein sequence of the OPRTase of S. cerevisiae from the DNA sequence and compared it to that of Escherichia coli, Podospora anserina and Dictyostelium discoideum. Some important similarities in the structure of these four proteins have been found. Finally, we have quantified the transcription of the URA5 gene in different physiological conditions and confirmed that it was not under the control of UTP or any intermediary product of the pathway.
Mol Gen Genet 1989 Feb
PMID:Structure and expression of the URA5 gene of Saccharomyces cerevisiae. 265 91

Detailed analysis of the kinetics of inhibition of E. coli RNA-polymerase-catalyzed synthesis of dinucleotide pppApU by 8-oxy-GTP and 8-Br-GTP on promoter A1 of the bacteriophage T7 delta D111 with an incomplete set of substrates was carried out. In accordance with the mathematical models obtained, we calculated quantitative parameters of binding of these nucleotide analogs to the centers whose geometry is suitable for incorporation of ATP and UTP. 8-oxy-GTP and 8-Br-GTP compete with ATP for the binding center (their steady-state dissociation constant ratios are 2.1 and 2.4, respectively, whereas the constant for ATP is 0.3 mM) but, unlike ATP, they are not incorporated into the product. 8-oxy-GTP competes also with UTP (its steady-state dissociation constant ratio is 21.6, the constant for UTP is 0.03 mM). 8-Br-GTP does not interact with the binding center of UTP.
Mol Biol (Mosk)
PMID:[Kinetics of inhibition by 8-oxy-GTP and 8-bromo-GTP of Escherichia coli RNA-polymerase synthesis of pppApU dinucleotide on the promotor a1 of phage T7deltaD111 DNA in a limited set of substrates]. 267 76

To determine whether 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] regulates transcription of the rat renal calbindin-D28k gene, the rate of calbindin-D28k mRNA synthesis was measured directly in nuclei using the in vitro nuclear transcription assay. Nuclei were prepared from kidneys of vitamin D-deficient rats at various times after a single ip injection of 1,25-(OH)2D3, and transcription was allowed to proceed in vitro in the presence of [32P]UTP for 30 min at 29 C, at which time the incorporation of UTP into trichloroacetic acid-precipitable material was optimal. Incorporation of UTP was decreased by 64.6% by alpha-amanitin, which selectively inhibits polymerase II. Purified [32P]RNA was analyzed for newly synthesized calbindin-D-28k gene transcripts by hybridization to calbindin-D28k cDNA immobilized on nitrocellulose filters. Using this assay we found that the first significant increase in calbindin-D28k gene transcription occurred at 1 h, and the peak of transcriptional activity occurred at 2 h. Within 12 h of 1,25-(OH)2D3 treatment, calbindin-D28k gene transcription returned to control levels. Using Northern blot analysis, a significant increase in calbindin-D RNA was first observed 2 h after hormone administration, reaching a maximum at 12 h. Renal calbindin-D28k protein levels are significantly increased by 3 h and reach a maximum value 48 h after hormone administration. Our results suggest that the early increase in renal calbindin-D28k may be due to transcriptional regulation. The long time lag between transcription and the peak of calbindin mRNA and calbindin protein accumulation may reflect the involvement of post-transcriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1989 Mar
PMID:Transcriptional regulation and chromosomal assignment of the mammalian calbindin-D28k gene. 274 55

The phosphorylation of deoxyguanosine was measured in fractured and intact mitochondria and an apparent Km of 16 microM for deoxyguanosine was calculated using fractured mitochondria. The effects of various deoxynucleotides on the phosphorylating activity in fractured organelles was tested at both a high and low ratio of NXP/ATP and at two pH values, 7.0 and 5.5. Exogenous dGTP, dGDP or dITP were inhibitory under all conditions tested. With a NXP/ATP ratio of 0.08 at pH 7.0, TTP, TDP, dADP, ADP, UTP and UDP were stimulatory, but at pH 5.5 only TTP elicited that response. When the NXP/ATP ratio was 10 at pH 5.5, TTP and UTP increased the activity more than 10-fold, whereas, at pH 7.0 TTP, TDP, dADP, ADP, UTP, UDP caused stimulation, but to a much lesser extent. When exogenous Mg2+, Mn2+ or Ca2+ were added to intact mitochondria, the rates of phosphorylation were lowered. In fractured mitochondria in the absence of exogenous ATP, little phosphorylation occurs, hence these metal ions caused little change. ATP-Mg, ATP-Mn and ATP-Ca, each at 0.05 mM caused a small inhibition with intact mitochondria, whereas, these compounds supported phosphorylation with fractured organelles. ATP-Mn (10 mM) or ATP-Ca (10 mM) stimulated phosphorylation in both intact and fractured mitochondria. Intact mitochondria synthesized dGMP, dGDP and dGTP when metal ion or ATP-Me concentrations were low (0.05 mM) or when Mg2+ concentration was high (10 mM). Additions of ATP-Ca, ATP-Mn, ATP-Mg, Mn2+ or Ca2+ at 10 mM cause the loss of dGDP and dGTP formation and, in most cases, an increase in the synthesis of dGMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biochem 1987 Oct
PMID:Phosphorylation of deoxyguanosine in intact and fractured mitochondria. 283 Apr 81

The effects of the inhibitors of IMP dehydrogenase, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and mycophenolic acid, on the synthesis of cellular glycoproteins were evaluated in Sarcoma 180 cells. Both tiazofurin and mycophenolic acid decreased the rate of incorporation of [2-3H]mannose and [2-3H]fucose into acid-precipitable glycoproteins within 4 hr of exposure; this inhibitory activity was concentration dependent and occurred in the absence of a significant effect on the incorporation of labeled glucosamine and leucine into acid-insoluble material. Interference with the utilization of [3H]mannose for the formation of glycoproteins was paralleled by an inhibition of [3H] mannose incorporation into their lipid-linked oligosaccharide precursors following treatment with cytotoxic concentrations of tiazofurin (100 microM) or mycophenolic acid (10 microM); these actions occurred within 3 hr of exposure to these agents, with maximal reductions being observed at 12 hr. Under these conditions, intracellular GTP levels were reduced by 80%, whereas ATP pools remained unaffected ant UTP levels were markedly increased. Guanosine (100 microM) prevented the cytotoxic actions of tiazofurin and mycophenolic acid and reversed the drug-induced decrease in GTP pools and in the incorporation of mannose and other metabolic precursors into acid-insoluble material. Inhibition of fucose and mannose incorporation into lipid-linked oligosaccharides and glycoproteins were preceded by decreases in the labeling of their respective guanosine nucleotide sugars and were followed sequentially by alterations in the plasma membrane as detected by both the binding and the rate of cell agglutination caused by the plant lectin, concanavalin A. The findings that tiazofurin and mycophenolic acid produce alterations in the utilization of [3H]mannose for the formation of glycoproteins and in membrane architecture are indicative of metabolic lesions induced by agents that selectively depress guanine nucleotide synthesis through inhibition of IMP dehydrogenase.
Mol Pharmacol 1985 Dec
PMID:Effects of the inhibitors of IMP dehydrogenase, tiazofurin and mycophenolic acid, on glycoprotein metabolism. 286 64

Evidence has been obtained for the presence of enzymes of both the de novo and salvage pyrimidine pathways in the protozoan parasite, Crithidia luciliae. Carbamyl phosphate synthetase-II activity could not be unequivocally demonstrated in crude extracts. However, a distinct peak of activity with a molecular weight of approximately 500 000 was observed following chromatography on Sepharose CL-6B. The enzyme preferentially utilised glutamine with respect to ammonia. It was inhibited by UTP and 5-phosphoribosyl-1-diphosphate had a small activating effect. Carbamyl phosphate synthesis by a 'phosphorolytic' citrullinase could not be demonstrated. The ensuing three de novo enzymes could also be separated on Sepharose CL-6B. Approximate molecular weights were estimated: aspartate transcarbamylase (150,000); dihydroorotase (90,000) and dihydroorotate dehydrogenase (70,000). As reported previously, orotate phosphoribosyltransferase and orotidylate decarboxylase were particulate, being associated with the glucosome. Activities of the salvage enzymes, uracil phosphoribosyltransferase, uridine phosphorylase and uridine nucleosidase were observed. All enzymes were cytoplasmic. No uridine kinase activity was detected.
Mol Biochem Parasitol 1986 May
PMID:Enzymes of pyrimidine biosynthesis in Crithidia luciliae. 287 7

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS-) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [3H]uridine and [3H]thymidine. The CTPS- mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS- cells are deficient in CTP synthetase and thus are unable to convert [3H]UTP into [3H]CTP, which eventually is converted into [3H]dCTP and incorporated into DNA; (2) the growth of CTPS- mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [3H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 x 10(-7). The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [3H]uridine labeling assay. This modified "tritium suicide" method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement.
Somat Cell Mol Genet 1989 Jan
PMID:Single-step selection of mammalian cell mutants deficient in CTP synthetase. 291 65

The Ca2+/Mg2+ ATPase of the rat heart sarcolemmal particles was solubilized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in non-denaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240,000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12-0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca2+ (Ka = 0.4 mM) and Mg2+ (Ka = 0.2 mM) as well as by other cations in the order Ca2+ greater than Mg2+ greater than Mn2+ greater than Sr2+ greater than Ba2+ greater than Ni2+ greater than Cu2+. The ATPase activity in the presence of Ca2+ was markedly inhibited by Mg2+, Mn2+, Ni2+ and Cu2+ whereas the monovalent cations such as Na+ and K+ were without effect. The enzyme did not exhibit Ca2+ stimulated Mg2+ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca2+ or Mg2+ ATPase activity was 8.5-9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca2+ metabolism in the myocardium is discussed.
Mol Cell Biochem 1988 May
PMID:Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma. 297 73


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