Isolation and initial characterization of the single polypeptide that ssynthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase

McClard, Ronald W.; Black, Michael J.; Livingstone, Laura R.; Jones, Mary Ellen

doi:10.1021/bi00561a024

Cited by 86 publications

(30 citation statements)

References 38 publications

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“…1). In animals (21) and plants (1,14,37), both enzymic activities reside on a single UMP synthase polypeptide.…”

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confidence: 99%

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Santoso¹,

Thornburg²

1992

Plant Physiol.

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Uridine 5'-monophosphate (UMP) synthase mutants of tobacco have been produced from haploid cell-suspension cultures of a transgenic Nicotiana tabacum line, Tr25. The mutants were induced by incubating the suspension-cultured cells with 1 mm N-nitroso-N-methylurea for either 5 or 12 hours. Twenty mutant calli were isolated on selection medium containing 20 milligrams per liter of 5-fluoroorotic acid. Of those tested, most had reduced regeneration capacity. Characterization of UMP synthase activities in the isolated calli showed that UMP synthase activity varied from 8 to nearly 100% of the wild-type activity. The growth of the calli on the media containing different levels of 5-fluoroorotic acid correlated with decreasing UMP synthase activity. Because the UMP synthase enzyme has two separate enzymic activities (orotate phosphoribosyl transferase and orotidine-5'-monophosphate decarboxylase), several mutants were further characterized to determine how the mutations affected each of the two enzymic activities. In each case, the enzymic activity affected was the orotate phosphoribosyl transferase and not the orotidine-5'-monophosphate decarboxylase. The wound-inducible phenotype of the Tr25 plants as measured by the activation of the pin2-CAT gene remained unchanged by introduction of the UMP synthase mutations. studying wound-inducible genes in the Solanaceae. In this paper we describe the first step in such a selection scheme, the production and characterization of mutants that have reduced levels of UMP synthase. These mutants will be used in the future for selection of mutants that are blocked in the wound-induction pathway. In addition, these mutants should also be useful to understand the biosynthesis of pyrimidines in plants.

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“…1). In animals (21) and plants (1,14,37), both enzymic activities reside on a single UMP synthase polypeptide.…”

mentioning

confidence: 99%

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Santoso¹,

Thornburg²

1992

Plant Physiol.

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“…Although the drug may eventually be incorporated into T. foetus RNA to slow the growth of the organism (because it is converted to 5-fluorouridine triphosphate in T. foetus), its inhibition of incorporation of uracil, uridine, and cytidine also emphasizes the importance of a functioning uracil phosphoribosyltransferase for T.foetus. This enzyme may have unique properties because it has no orotate phosphoribosyltransferase activity and has not been found in most mammalian tissues (18 …”

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Pyrimidine metabolism in Tritrichomonas foetus.

Wang

Verham

Tzeng

et al. 1983

Proc. Natl. Acad. Sci. U.S.A.

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The anaerobic parasitic protozoa Tritrichomonas foetus is found incapable of de novo pyrimidine biosynthesis by its failure to incorporate bicarbonate, aspartate, or orotate into pyrimidine nucleotides or nucleic acids. Uracil phosphoribosyltransferase in the cytoplasm provides the major pyrimidine salvage for the parasite. Exogenous uridine and cytidine are mostly converted to uracil by uridine phosphorylase and cytidine deaminase in T.foetu prior to incorporation. T. foetus cannot incorporate labels from exogenous uracil or uridine into DNA; it has no detectable dihydrofolate reductase or thymidylate synthetase and is resistant to methotrexate, pyrimethamine, trimethoprim, and 5-bromovinyldeoxyuridine at millimolar concentrations. It has an enzyme thymidine phosphotransferase in cellular fraction pelleting at 100,000 x g that can convert exogenous thymidine to TMP via a phosphate donor such as p-nitrophenyl phosphate or nucleoside 5'-monophosphate. Thymidine salvage in T. foetus is thus totally dissociated from other pyrimidine salvage.It has become apparent in recent years that parasitic protozoa are generally incapable of de novo synthesis of purine nucleotides. Trypanosoma cruzi (1), Leishmania donovani (2), Plasmodium lophurae (3), Eimeria tenella (4), and Trichomonas vaginalis (5), to name but a few examples, depend on specific networks of salvage pathways to fulfill their purine requirements. Because of this deficiency in metabolic activities, rational approaches to controlling some of the parasites have been possible. Allopurinol exhibits antitrypanosomal and antileishmanial activities because it is recognized by the parasite salvage enzymes as a hypoxanthine analog (6, 7). Allopurinol riboside (8), formycin B (9), and 4-thiopyrazolopyrimidine riboside (10) have antileishmanial activities because of the nucleoside phosphotransferase in leishmania, which incorporates the compounds into parasite's nucleotide pool.De novo pyrimidine biosynthesis, on the other hand, takes place in most of the parasitic protozoa. Recently, it has been reported that the anaerobic flagellates Trichomonas vaginalis and Giardia lamblia may not, however, have even the capability of pyrimidine de novo synthesis. The former lacks aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase, and orotate phosphoribosyltransferase in its crude extract (11), whereas the latter indicates no incorporation of aspartate into the cold trichloroacetic acid-insoluble fraction (12). These results suggest that anaerobic flagellates may differ from other protozoan parasites in lacking both purine and pyrimidine de novo synthetic abilities and thus may offer even more opportunities for chemotherapeutic attack.To verify these possibilities, we studied pyrimidine metabolism in Tritrichomonasfoetus, a Precursor Incorporation into the Nucleotide Pool. T. foetus cells were washed, suspended in phosphate-buffered saline, pH 7.2 (Pi/NaCl)/20 mM glucose to a final cell density of 108/ml, and incubated at 370C. A radiolabeled subst...

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“…Cells in culture that have developed resistance to 6azauridine or pyrazofurin, inhibitors of ODC activity, have coordinately increased levels of both OPRT and ODC activities (8,9), indicating that the activities are closely linked. A single protein of M, =51,500 that contains both the OPRT and ODC activities has now been purified from mouse ascites cells (10) and from human placenta (11). This bifunctional protein was designated UMP synthase.…”

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confidence: 99%

Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase.

Suttle

Bugg

Winkler

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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The last two steps in the de novo biosynthesis (20). Positive plaques were isolated through two rounds of selection. The UMP synthasespecific inserts were isolated from these positive recombinant plaques by EcoRI digestion and subcloned into the EcoRI site of pBR322 for further sizing and restriction map analysis. For isolation of UMP synthase genomic fragments, a human genomic library prepared in the A Charon 4A vector (21) was screened with the human UMP synthase-specific plasmid pHUSc22 (17) as described for the cDNA libraries.DNA Sequencing. Specific restriction fragments of the UMP synthase inserts were isolated from low-meltingtemperature agarose and subcloned into M13 cloningsequencing vectors mp8/mp9 (22) or mpl8/mpl9 (23). The sequence of the fragments was determined by the dideoxy Abbreviations: QDC, orotidine-5'-monophosphate decarboxylase;

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Isolation and initial characterization of the single polypeptide that ssynthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase

Cited by 86 publications

References 38 publications

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Isolation and Characterization of UMP Synthase Mutants from Haploid Cell Suspensions of Nicotiana tabacum

Pyrimidine metabolism in Tritrichomonas foetus.

Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase.

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