Increased β‐aminoisobutyric acid in rat liver with 6‐azauracil and its enantiomer

Tamaki, Nanaya; Fujimoto, Shigeyoshi; Mizutani, Naomi; Mizota, Chizuru

doi:10.1016/0014-5793(85)81004-8

Cited by 7 publications

(5 citation statements)

References 17 publications

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“…Moreover, the R isomer of P-aminoisobutyric acid accumulates in the liver of rats treated with 6-azauracil [41] which is a metabolic inhibitor of amino acid aminotransferase. On the other hand, pyrimidine is reduced by dihydropyrimidine dehydrogenase with an overall truns-addition of hydrogen at the si-face of C5 and C6 [42,431.…”

Section: Discussionmentioning

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: Discussionmentioning

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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Section: D-3-aminoisobutyrate -Pyruvate Aminotransferase [(R)-mentioning

confidence: 95%

“…AlaAT-I1 (K, = 0.81 mM) was higher than to AlaAT-I (K, = 6.3 mM). The concentration of j-alanine in rat liver is 0.180 0.028 pmol/g wet tissue [24]. Therefore, AlaAT-I1 also has an important role in p-alanine metabolism.…”

Section: Inhibition Of Alaat-ii By Uracil Analogsmentioning

confidence: 96%

“…Moreover, when 6-azauracil was injected subcutaneously, 3-aminoisobutyric acid was also increased 22-fold in rat liver [24]. The incorporation of [rnethyl-'4C]thymine into 3-aminoisobutyric acid was increased 42-fold by 6-azauracil [24]. 6-Azauracil was a noncompetitive inhibitor of purified AlaAT-I from rabbit liver [25], but the mode of action of 6-azauracil on AlaAT-I1 is obscure.…”

Section: D-3-aminoisobutyrate -Pyruvate Aminotransferase [(R)-mentioning

confidence: 98%

“…Therefore, 6-azauracil cannot be classified in the group of carbonyl agents such as aminooxyacetic acid. These results might cause the accumulation of D-3-aminoisobutyric acid as well as p-alanine in the liver of rats administered 6-azauracil subcutaneously [24]. Gabaculine, 5-amino-I ,3-cyclohexadienylcarboxylic acid, is a very efficient enzyme-activated inhibitor of 4-aminobutyrate aminotransferase [33 -361.…”

Section: Kinetics Of 6-azauracil Toward Alaat-iimentioning

confidence: 99%

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Purification, characterization and inhibition of d‐3‐aminoisobutyrate aminotransferase from the rat liver

Tamaki

Kaneko

Mizota

et al. 1990

European Journal of Biochemistry

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D-3-Aminoisobutyrate -pyruvate aminotransferase was purified 2000-fold from rat liver extract using heat treatment, ammonium sulfate fractionation, carboxylmethyl-Sepharose CL-6B, DEAE-Sepharose CL-6B, hydroxyapatite, Sephacryl S-200 and electrofocusing chromatographies. 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 220 kDa and the subunit molecular mass was 52 kDa. The enzyme exhibited absorption maxima at 280 nm and 412 nm with a shoulder at 330 nm at neutral pH. The pH optimum for enzyme activity was 9.5 and the K, values for p-alanine and pyruvic acid were calculated to be 0.81 mM and 0.45 mM, respectively. The purified enzyme catalyzed the transamination of w-amino acids; P-alanine and D-3-aminoisobutyric acid served as good amino donors, and pyruvic acid, glyoxylic acid and oxaloacetic acid were favorable amino acceptors.6-Azauracil and 6-azathymine were found to be potent inhibitors of purified rat liver D-3-aminoisobutyratepyruvate aminotransferase. 6-Azauracil acted as a competitive inhibitor with respect to p-alanine, and was an uncompetitive inhibitor with respect to pyruvic acid with a Ki of approximately 8.9 mM.

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Identity of D-3-aminoisobutyrate-pyruvate aminotransferase with alanine-glyoxylate aminotransferase 2

Kontani

Kaneko

Kikugawa

et al. 1993

Biochimica et Biophysica Acta (BBA) - General Subjects

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Increased β‐aminoisobutyric acid in rat liver with 6‐azauracil and its enantiomer

Cited by 7 publications

References 17 publications

Purification, characterization and inhibition of dihydropyrimidinase from rat liver

Purification, characterization and inhibition of dihydropyrimidinase from rat liver

Purification, characterization and inhibition of d‐3‐aminoisobutyrate aminotransferase from the rat liver

Identity of D-3-aminoisobutyrate-pyruvate aminotransferase with alanine-glyoxylate aminotransferase 2

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