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The in vitro inhibition of purine biosynthesis de novo by a series of cyanopyrazoles was studied. At concentration 1 mM trichloromethyl analogs (3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole and N-hydroxyethyl-3(5)-amino-4-cyano-5(3)-trichloromethylpyrazole) were found to inhibit IMP synthesis 80 and 30% respectively. GAR synthesis was inhibited at a lower degree at the same range of concentrations. The compounds demonstrated a similar pattern of inhibition of the last steps, e.g. AICAR formylation and cyclization as found on the whole pathway.  相似文献   

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The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.  相似文献   

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With the emergence of multifaceted bioinformatics-derived data, it is becoming possible to merge biochemical and physiological information to develop a new level of understanding of the metabolic complexity of the cell. The biosynthetic pathway of de novo pyrimidine nucleotide metabolism is an essential capability of all free-living cells, and it occupies a pivotal position relative to metabolic processes that are involved in the macromolecular synthesis of DNA, RNA and proteins, as well as energy production and cell division. This regulatory network in all enteric bacteria involves genetic, allosteric, and physiological control systems that need to be integrated into a coordinated set of metabolic checks and balances. Allosterically regulated pathways constitute an exciting and challenging biosynthetic system to be approached from a mathematical perspective. However, to date, a mathematical model quantifying the contribution of allostery in controlling the dynamics of metabolic pathways has not been proposed. In this study, a direct, rigorous mathematical model of the de novo biosynthesis of pyrimidine nucleotides is presented. We corroborate the simulations with experimental data available in the literature and validate it with derepression experiments done in our laboratory. The model is able to faithfully represent the dynamic changes in the intracellular nucleotide pools that occur during metabolic transitions of the de novo pyrimidine biosynthetic pathway and represents a step forward in understanding the role of allosteric regulation in metabolic control.  相似文献   

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A mutant of Brevibacterium ammoniagenes producing large quantities of UMP and uracil is described. The mutations render bacteria braditrophic for arginine, sensitive to adenine, resistant to rifampicin and pyrimidine analogues 5-fluorouracil, 5-fluorouridine, azauracil and thiouracil. The activities of enzymes involved in the UMP biosynthesis, i.e. orotate phosphoribosyltransferase, orotate-5-monophosphate decarboxylase, dihydroorotate oxidase, are 4-, 3.5- and 4.5-fold higher in the mutant than in the parent strain when grown in minimal medium. The synthesis of these enzymes in mutant cells is not repressed in the presence of exogenous Ura. True revertants, which completely restore the wild-type phenotype, occur among the Arg+ clones. The nature of the mutation is discussed.  相似文献   

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A sensitive radioassay is described for the overall biosynthetic activity of the multienzymatic protein which catalyzes the first three reactions of de novo pyrimidine biosynthesis in mammals. The ability of the multienzymatic protein to synthesize dihydroorotate can be assayed using [14C]HCO3?, l-[14C]aspartate, or [14C] carbamyl phosphate as substrate. The synthesis of the final product, l-dihydroorotate, may be coupled to synthesis of orotidine 5′-monophosphate to overcome the unfavorable equilibrium existing between l-dihydroorotate and its precursor, N-carbamyl-l-aspartate, in the physiological pH range (Christopherson, R. I., and Jones, M. E., 1979, J. Biol. Chem.254, 12506–12512). l-Aspartate and all pyrimidine intermediates from carbamyl phosphate to orotidine 5′-monophosphate can be clearly separated by ion-exchange chromatography in a single dimension on polyethyleneimine-cellulose chromatograms and carbamyl phosphate and its degradation product cyanate may be quantitated directly along with the other intermediates.  相似文献   

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Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.  相似文献   

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Gas chromatographic/mass spectrometric methods for the measurement of the flux through the de novo pyrimidine biosynthetic pathway by quantitating the incorporation of [13C]bicarbonate and 13CO2 into the uracil nucleotide pool in L1210 tumors are reported. Simultaneous measurements of the incorporation of [13C]bicarbonate and the more commonly used [14C]bicarbonate into uridine of L1210 cells in vitro showed that the two methods were comparable. A modification of the method was applied to in vivo studies where the incorporation of 13CO2 into the uracil nucleotide pool of L1210 tumors in mice was quantitated. The measurements were used to determine changes in the flux through the de novo pyrimidine pathway in animals pretreated with known inhibitors of the pathway. A comparison of control animals with those pretreated with 6-azauridine, acivicin, and pyrazofurin resulted in mean percentage inhibitions of 87, 95, and 94%, respectively. This technique should allow investigation of the respective contributions of salvage and de novo synthesis in the formation of pyrimidines in vivo and the effects of agents designed as enzyme inhibitors of the de novo pathway.  相似文献   

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The subcellular distribution of the enzymes of de novo pyrimidine nucleotide biosynthesis was investigated in pea (Pisum sativum L. cv Progress No. 9) leaves. Aspartate carbamoyltransferase, the committed step of the pathway, was found to be strictly confined to the chloroplasts. Dihydro-orotase, orotate phosphoribosyl transferase, and orotidine decarboxylase activities were also found only in the plastids. The remaining enzyme of the pathway, dihydroorotate dehydrogenase, was shown to be mitochondrial.  相似文献   

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The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects.  相似文献   

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Effect of sodium butyrate on DNA synthesis and the induction of ornithine decarboxylase (EC 4.1.1.17), a rate-limiting enzyme of polyamine biosynthesis, was studied in phytohemagglutinin(PHA)-stimulated bovine lymphocytes. Millimolar concentrations of butyrate completely inhibited the incorporation of [3H] thymidine into the acid-insoluble fraction and reversibly suppressed the induction of ornithine decarboxylase. Other shortchain fatty acids were much less active than butyrate. These results suggest that the suppression of ornithine decarboxylase activity may be one of the reasons for the inhibition of DNA synthesis with butyrate in bovine lymphocytes, because our previous experimental results have shown that the induction of ornithine decarboxylase closely correlates with the DNA synthesis in growth-stimulated cells.  相似文献   

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The isolation and characterization of a new mutant of Chinese hamster ovary cells is described. This mutant, Urd-A, shows an absolute requirement for exogenously added pyrimidines for growth. Complementation analysis indicates that the lesion in this mutant is recessive. Revertants can be isolated at frequencies suggesting that it is a single gene alteration. Biochemical analysis of cell-free extracts of CHO-K1 (Urd+) and Urd-A revealed that Urd-A possesses no more than 10% of wild-type levels of carbamyl phosphate synthetase (EC 2.7.2.9) activity, no more than 1% of wild-type levels of aspartate transcarbamylase (EC 1.2.3.2) activity, and undetectable levels of dihydroorotase (EC 3.5.2.3) activity. Thus, this mutant appears simultaneously to possess marked or complete deficiencies in the activities of the first three enzymes of pyrimidine biosynthesis. Activities of the other enzymes of the pathway appear normal. The use of this mutant for biochemical-genetic studies of pyrimidine biosynthesis is discussed.  相似文献   

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