Chromatography of Pyrimidine Reduction Products

Fink, Robert M.; Cline, Richard E.; McGaughey, C.A.; Fink, Kay

doi:10.1021/ac60109a003

Cited by 147 publications

(49 citation statements)

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“…Carboxymethyl-Sepharose CL-6B, DEAE-Sepharose CL-6B, and Sephacryl S-300 were obtained from Pharmacia and hydroxyapatite from Nihon Chemical Ltd (Tokyo). 5-Bromo-5,6-dihydrouracil (BrH,Ura) and N-carbamoyl-DL-Paminoisobutyric acid were synthesized according to ZeeCheng et al 1231 and Fink et al [24], respectively.…”

Section: Methodsmentioning

confidence: 99%

Purification, characterization and inhibition of dihydropyrimidinase from rat liver

Kikugawa

Kaneko

Fujimoto-Sakata

et al. 1994

European Journal of Biochemistry

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Dihydropyrimidinase (DHPase) was purified 564-fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE-Sepharose CL-6 B, carboxymethyl-Sepharose CLQB, hydroxyapatite and Sephacryl S-300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6-dihydrouracil (HJJra) to N-carbamoyl-P-alanine or 5,6-dihydrothymine (H,Thy) to N-carbamoyl-P-aminoisobutync acid.Authentic 5-bromo-5,6-dihydrouracil (BrH,Ura) and commercially available H,Thy were racemic. However, these 5-substituted 5,6-dihydropyrimidines were hydrolyzed by over 96% and 9 8%, respectively, by DHPase. These results suggest that dihydropyrimidinase has no stereo specificities for 5-substituents of H,Ura. The addition of H,Ura and H,Thy competitively inhibited the enzyme activity against BrH,Ura. However, the addition of N-carbamoyl-P-alanine or N-carbamoyl-j?-aminoisobutyric acid showed hyperbolic mixed-type inhibition, when BrH,Ura was used as the substrate. The values of the dissociation constants of BrH,Ura, N-carbamoyl-P-alanine and N-carbamoyl-Paminoisobutyric acid were 17 pM, 0.38 mh4 and 0.38 mM, respectively.DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atomhubunit. Zn2+ also inhibited the hydrolysis of BrH,Ura by the enzyme. The K, for Zn" as an inhibitor of DHPase was 23 pM, and the maximum rate of inactivation was 0.057 min-' at 37OC. HJJra and H,Thy protected the enzyme activity from Zn2+ inactivation.Uracil is metabolized to P-alanine via 5,6-dihydrouracil (HJJra) and N-carbamoyl-P-alanine [l, 21. The enzymes of uracil catabolism are also active toward thymine, metabolizing it via dihydrothymine (H,Thy) and N-carbamoyl-Paminoisobutyric acid to (R)-P-aminoisobutyric acid [l, 3, 41. In mammals, P-alanine and (R)-P-aminoisobutyric acid are transported into mitochondria [5, 61, where they are further metabolized to acetyl-CoA and propionyl-CoA, respectively, by P-alanine-oxoglutarate aminotransferase, (R)-P-aminoCorrespondence to N. Tamaki, Laboratory of Nutritional Chemistry, Faculty of Nutrition, Kobe-Chin University, Nishi-ku, Kobe 651-21, JapanAbbreviations. DHPase, dihydropyrimidinase ; BrH,Ura, 5-bromo-5,6-dihydrouil: H,Ura, 5,6-dihydrouracil; H,Thy, 5,6-dihydrothymine.Enzymes. Dihydropyrimidinase, 5,6-dihydropyrimidine amidohydrolase (EC 3.5.2.2); 4-aminobutyrate aminotransferase, P-alanine-oxoglutarate aminotransferase (EC 2.6.1.19) ; (R)-3-amino-2-methylpropionate-pyruvate aminotransferase, ~-3-aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40) ; methylmalonate semialdehyde dehydrogenase (EC 1.2.1.27) ; dihydropyrimidine dehydrogenase (EC 1.3.1 .l); carbamoyl-phosphate synthetase (EC 6.3.5.5) ; aspartate transcarbamoylase (EC 2.1.3.2) ; dihydroorotase (EC 3.5.2.3); RNA nucleotidyltransferase (EC 2.7.7.6) ; pyruvate kinase (EC 2.7.1.40); lactate dehydroge...

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Section: Methodsmentioning

confidence: 99%

Purification, characterization and inhibition of dihydropyrimidinase from rat liver

Kikugawa

Kaneko

Fujimoto-Sakata

et al. 1994

European Journal of Biochemistry

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“…The dihydrothymine derivatives urea, and substituted ureas were detected by spraying the chromatograms with 0.5 N aqueous sodium hydroxide, air drying for 30 min, and respraying with a solution ofpdimethylaminobenzaldehyde (PDAB) (1 g in 100 ml 95% ethanol plus 10 ml concentrated hydrochloric acid), after the method of Fink et a/. (19). Iodine vapor was used to detect products on silica gel plates.…”

Section: Methodsmentioning

confidence: 99%

The Ultrasonic Degradation of Thymine

Mead¹,

Sutherland²,

Verrall³

1975

Can. J. Chem.

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Chemical reactions that occur in liquids subjected to ultrasonic irradiation (insonation) of sufficient intensity to induce cavitation, have been attributed (1) to a number of processes; the reactive species produced in water and aqueous solutions during cavitation are hydroxyl radicals (2), hydrated electrons (3), hydroperoxyl radicals (4), and hydrogen atoms (5). These species are also produced during the radiolysis of water and in this paper we show that the sonolysis of thymine may be correlated with its radiolytic degradation.The radiolysis of aqueous thymine solutions has been the subject of numerous investigations and studies by Teoule and co-workers (6-11) identified more than 26 products formed during 60Co-y irradiation. Hydroxyl radicals produced by ionizing radiation react with thymine to give 6-hydroxy-5,6-dihydro-5-thyml and 5-hydroxy-5,6-dihydro-6-thymyl radicals; hydrogen atoms (or e,,-+ H,O+) react with thymine to give (5-uracyl)methyl, 5,6-dihydro-5-thyml, and 5,6-dihydro-6-thyml radicals (1 1). These radicals react. very rapidly with dissolved oxygen (e.g. K(TOH + 0,) = 1.5 x lo9 M -,l S-l (12) to produce hydroperoxide radicals which are reduced to the hydroperoxyhydroxydihydro products ( Fig. 1) (5 and 6) and the hydroperoxide products (7, 5-hydroperoxy-5,6-dihydrothymine, cis-and trans-6-hydroperoxy-5,6-dihydrothy-'For a preliminary communication see ref. 27. mine). The peroxide products are relatively unstable (11) and decompose to give various alcohols (cis-and trans-9, 8, 5-hydroxy-5,6-dihydrothymine, cis-and trans-6-hydroxy-5,6-dihydrothymine). In addition, the hydroxyhydroperoxides decompose to give 5-hydroxy-5-methylbarbituric acid and N-formyl-N'-pyruvylurea and the remaining products result from decomposition of N-formyl-N'-pyruvylurea to give pyruvylurea, urea, and formylurea.El'piner (1) was the first to investigate the effect of ultrasound on purine and pyrimidine bases. He insonated (500 kHz) aqueous solutions of the bases at an intensity of 5 W cm-2 in the presence of various gases. In the U.V. spectra of the insonated bases, the absorption maxima were reduced and the minima shifted to longer wavelengths. Uracil was found to be the most sensitive, adenine and guanine less so. In a later study, El'piner and Sokol'skaya (13) insonated aqueous solutions of cytosine, uracil, thymine, and adenine and investigated the products of sonolysis chromatographically but no degradation products were identified. The reduction in intensity of the U.V. absorption band of the various purines and pyrimidines was attributed to ring cleavage (1). Since the effect was considerably reduced in the presence of gases other than oxygen, it was suggested that molecular oxygen played an important role and a smaller oxidative role was assigned to hydroxyl radicals.This study was undertaken to reinvestigate the sonolysis of aerated aqueous thymine solutions Can. J. Chem. Downloaded from www.nrcresearchpress.com by 34.210.69.67 on 05/11/18For personal use only.

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“…BUIB was prepared from DHT and dilute alkali, and both compounds were identified by spraying the dried chromatogram with dilute alkali followed by p-dimethylaminobenzaldehyde (15 No attempt was made to purify the inhibitory factor extensively, but when prepared from normal leukocytes, it was found in the precipitate after 40%o saturation with ammonium sulfate. It was not dialyzable, and activity was lost after heating to 600 C for 30 minutes.…”

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confidence: 99%