Free Uracil Research Articles

An N -Glycosidase from Escherichia coli That Releases Free Uracil from DNA Containing Deaminated Cytosine Residues

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg(++) has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.

Harnstoff-Metabolismus bei Basidiomyceten

Summary In fruit-bodies of the commercial mushroom, Agaricus bisporus L ange , uracil is metabolized both in anabolic and catabolic reactions. The main radioactive degradation product in uracil-2- 14 C feedings is urea. The relationship between pyrimidine breakdown and urea biosynthesis might be an indirect one. Uracil is obviously reductively degraded, resulting carbon dioxide is introduced into urea formation by way of the Krebs-Henseleit ornithine cycle. There is no evidence concerning any mode of oxidative pyrimidine catabolism. - Urea biosynthesis from uracil-2- 14 C in Agaricus caps is inhibited by pretreatment with DL- s -methylaspartate, hadacidine, DL- δ -Nacetylornithine, DL-N-allylasparate, or KF. In experimentes using NaH 14 CO 3 and L-citrullineureido- 14 C, radioactive citrulline accumulates after treatment with s-methylaspartate, suggesting inhibition of argininosuccinate synthesis. N-Allylaspartate and potassium fluoride may influence carbamylphosphate synthesis. KF quantitatively inhibits nucleotide synthesis from uracil. 14 CO 2 formed by reductive degradation of uracil-2- 14 C is only partly released. Most of the evolved carbon dioxide is captured and used in urea synthesis. 5-Fluorouracil-2- 14 C is incorporated into urea, too. Pretreatment of Agaricus caps with nonlabelled fluorouracil results in decreased incorporation of radioactivity from uracil-2- 14 C into urea, suggesting some mode of „substrate competition“ at the active site of dihydrouracil dehydrogenase. 5-Bromouracil and 5-nitrouracil may act in a similar manner. Thymine-2- 14 C feedings do not result in urea labelling, supposing a permeability barrier. 2-Thiouracil-2- 14 C is metabolized but does not result in urea labelling. 6-Azathymine is an effective inhibitor of urea biosynthesis from uracil-2- 14 C possibly by affecting dihydrouracil dehydrogenase. A crude enzyme was prepared from acetone dried fruit-bodies of Agaricus by phosphate buffer extraction possessing dihydrouracil dehydrogenase aktivity as revealed by optical methods. Incubation of the preparation with uracil-2- 14 C results in carbon-dioxide- 14 C evaluation. Part of the evolved 14 CO 2 is fixed in urea synthesis. Uracil is not used in anabolic reactions. The enzyme preparation must contain the complete enzyme systems of both the reductive pyrimidine catabolic path and of urea biosynthesis by way of the ornithine cycle reactions. - In fruit-bodies of Agaricus bisporus L ange , pyrimidines are presumably formed according to the wellknown orotic acid scheme of pyrimidine biosynthesis. Preliminary experiments show that carbamylaspartateureido- 14 C is introduced into orotic acid and not identified nucleotides. Orotic acid-6- 14 C and orotic acid-2- 14 C are converted into nucleotides and free uracil. In the presence of 6-azauracil known as inhibitor of orotidylic acid decarboxylase orotate accumulates. There is no evidence that the observed urea formation from carbamylaspartate or orotate is a result of reversibility of orotic acid synthesis in fruit-bodies of Agaricus bisporus L ange . Orotate-2- 14 C is more effective in urea labelling than orotic acid-6- 14 C that might be used in urea formation via s -alanine. - In fruitbodies of Lycoperdon perlatum P ers . ( Lycoperdaceae ) orotic acid-6- 14 C gives rise to labelled s -alanine, suggesting the operation of the usual reductive pyrimidine catabolism. s -Alanine is not formed by aspartic acid decarboxylation. - In fruit-bodies of Panus tigrinus (Fr.) S ing . ( Tricholomataceae ) supplied pyrimidines are metabolized in a similar manner as in sporophores of Agaricus and Lycoperdon . Urea formation from pyrimidines, however, is not detectable, for an active urease is present.

The Formation and Metabolic Interchanges of Pseudouridine in Tetrahymena pyriformis

Abstract The formation and metabolism of pseudouridine (5-ribosyluracil) in growing Tetrahymena pyriformis have been studied with the aid of labeled uracil, uridine, pseudouridine, and glucose as precursors. The pyrimidine rings of uracil, uridine, and pseudouridine serve equally well as precursors of the pyrimidine component of uridine and pseudouridine of ribonucleic acid. The ribosyl moiety of uridine labels the ribosyl portions of RNA uridine and RNA pseudouridine equally. Although the latter was not degraded to establish unequivocally that uridine ribosyl is present in pseudouridine ribosyl, the equivalence of specific activities and the evidence of the presence of precursor uridine ribosyl in the uridine of the RNA make it appear that the uridine-pseudouridine interconversion takes place by an intramolecular rearrangement and not principally through a free uracil or diribosyluracil intermediate. Although some transribosylation involving purine and pyrimidine ribosyl moieties and some formation of RNA pyrimidine ribosyl from glucose were observed, these appear to be of secondary importance. The data are consistent with, but do not prove, a rearrangement of pyrimidine nucleosides at the polynucleotide level.

Studies on the metabolism of thymine and 6-azathymine

The administration of 6-azathymine to the mouse led to the urinary elimination not only of free azathymine, but also of various metabolites of it. Of these metabolites, two have been identified: (a) the ribonucleoside, (b) a side-chain oxidation product of azathymine, tentatively identified as 5-hydroxymethyl-6-azauracil (6-hydroxymethyl as-triazine-3:5-(2H:4H)-dione). Other metabolic derivatives of azathymine were produced, but their nature has not been established conclusively. The administration of azathymine also resulted in the excretion of relatively large amounts of free uracil. Following the administration of 6-azathymine-5- 14C to mice, radioactivity was found in all tissues investigated, not only in the form of free azathymine, but also as metabolic derivatives. A study of the influence of mouse liver slices or homogenates on radioactive azathymine and thymine indicated that each of these compounds is converted to the corresponding ribonucleoside and, to a lesser degree, to the analogous 2'-deoxyribonucleoside.

Enzymes of uracil metabolism in the Ehrlich ascites tumour and mammalian liver

The content of enzymes involved in uracil metabolism (pyrimidine nucleoside phosphorylase, pyrimidine nucleoside kinase and enzymes of uracil catabolism) in extracts from rat liver, mouse liver and the Ehrlich ascites tumour was studied. Much higher levels of enzymes involved in anabolic reactions were found in ascites tumour extracts than in liver, whereas the enzymes of uracil catabolism which were present in rat and mouse liver were absent from the tumour. Especially the relatively high concentration of pyrimidine nucleoside kinase in the tumour extracts probably explains the better utilization of free uracil for polynucleotide synthesis in this tissue as compared to rat liver. Pyrimidine nucleoside kinase needed a high concentration of uridine ( ca. 0.01 M) for optimal synthesis of nucleosides, while a considerably lower concentration of cytidine (less than 0.0055 M) was sufficient. This partly explains the much better utilization of cytidine for polynucleotide synthesis as compared to uridine.

STUDIES IN PYRIMIDINE METABOLISM

By partial hydrolysis of yeast nucleic acid, preparations containing pyrimidines as the only nitrogenous constituents were prepared and administered to rabbits. Uracil nucleoside when administered per os, subcutaneously or intraperitoneally caused an increased excretion of urea often much more than enough to account for the nitrogen administered. The undetermined nitrogen (the difference between the total nitrogen and the urea nitrogen) was always increased. A part of the increase in the undetermined nitrogen was due to the excretion of free uracil which was isolated in pure crystalline form. As much as 20 per cent. of the uracil fed as the nucleoside was recovered free in the urine.When a mixture of cytosine and uracil nucleosides was administered to rabbits, there was an increased excretion of urea and usually no increase in the undetermined nitrogen. Uracil was isolated from the urine of one animal and was barely detected by a color reaction in another. No increase of creatine, creatinine, or purines w...

Studies in pyrimidine metabolism

By partial hydrolysis of yeast nucleic acid, preparations containing pyrimidines as the only nitrogenous constituents were prepared and administered to rabbits. Uracil nucleoside when administered per os, subcutaneously or intraperitoneally caused an increased excretion of urea often much more than enough to account for the nitrogen administered. The undetermined nitrogen (the difference between the total nitrogen and the urea nitrogen) was always increased. A part of the increase in the undetermined nitrogen was due to the excretion of free uracil which was isolated in pure crystalline form. As much as 20 per cent. of the uracil fed as the nucleoside was recovered free in the urine. When a mixture of cytosine and uracil nucleosides was administered to rabbits, there was an increased excretion of urea and usually no increase in the undetermined nitrogen. Uracil was isolated from the urine of one animal and was barely detected by a color reaction in another. No increase of creatine, creatinine, or purines was detected after feeding either preparation. Not even a color reaction for pyrimidines was obtained by using the same procedures on the urine obtained after feeding yeast nucleic acid. Mendel and Myers were unable to find a trace of pyrimidine in the urine after feeding yeast nucleic acid but found that uracil, when fed, was excreted unchanged. Taken together, the data show that increasing quantities of uracil appear in the urine as simpler complexes containing the uracil group are fed. The conclusion may therefore be drawn (at least in respect to uracil), that, in the metabolism of yeast nucleic acid before the pyrimidine is liberated and even before the nucleoside is formed, the pyrimidine is altered in such a way that it may be further broken down and its nitrogen converted into urea.