Masae Kaneko

Molecular cloning and sequencing of a cDNA encoding dihydropyrimidinase from the rat liver

A cDNA encoding dihydropyrimidinase has been isolated from a rat cDNA library. The N-terminal and an internal amino acid sequences were determined, and PCR primers were designed based on these sequences. Using a cDNA fragment amplified by RT-PCR with these primers, three cDNA clones were isolated from a rat liver library. The clone with the longest insert of 2129 bp contained a 1557 bp open reading frame encoding a polypeptide of 519 residues with a molecular mass of 56,832 Da.

Analysis of cyclic feed intake in rats fed on a zinc-deficient diet and the level of dihydropyrimidinase (EC 3.5.2.2).

The body weight and feed intake of rats fed on a Zn-deficient diet for 28 d were reduced compared with those of control rats. The feed intakes of the Zn-deficient and control groups during the period were 10.2 (SE 0.3) and 15.7 (SE 0.2) g/d respectively. Cyclic variations in feed intake and body-weight changes were found in analysis not only of all the data for five rats but also that in each individual rat. Cosinor analysis revealed that the cyclical period of both the feed intake and body-weight change in the Zn-deficient rats was 3.5 (SE 0.1) d. The mesor and amplitude value of the feed intake in the Zn-deficient rats was 10.1 (SE 0.4) g/d and 3.5 (SE 0.5) g/d respectively, and that of body-weight change was 1.4 (SE 0.1) g/d and 7.9 (SE 1.3) g/d respectively. Among pyrimidine-catabolizing enzymes, dihydropyrimidinase (EC 3.5.2.2) activity showed significant retardation in the Zn-deficient rat liver with decrease of the enzyme protein. The ratio of apo-form to holo-form dihydropyrimidinase in the liver was not affected by the Zn-deficient diet.

Inhibition of d-3-aminoisobutyrate-pyruvate aminotransferase by 5-fluorouracil and α-fluoro-β-alanine

Among pyrimidine derivatives, we found that 5-fluorouracil;potently inhibited purified rat liver d -3-aminoisobutyrate-pyruvate aminotransferase, whereas 5-fluorouridine did so to a much lesser extent. 5-Fluorouracil acted as a competitive inhibitor against β-alanine with a K i of 56 μM, and was uncompetitive against pyruvic acid, with a K i of 73 μM. α-Fluoro-β-alanine, a metabolite of 5-fluorouracil, was also a competitive inhibitor with respect to β-alanine with a K i of 8.0 mM. 5-Fluorouracil acted also as a competitive inhibitor of 4-aminobutyrate aminotransferase with respect to β-alanine with a K i value of 1.9 mM, and was uncompetitive against 2-oxoglutaric acid, with a K i of 1.8 mM.

Evaluation of interconversion between (R)- and (S)-enantiomers of β-aminoisobutyrate

Abstract The conversion of (R)- to (S)-β-aminoisobutyrate was observed in the presence of d -3-aminoisobutyrate-pyruvate aminotransferase, aminobutyrate aminotransferase, pyruvate and l -glutamate. The reverse reaction was also found in the presence of 2-oxoglutarate and l -alanine. Neither d -3-aminoisobutyrate-pyruvate aminotransferase nor aminobutyrate aminotransferase revealed a racemase activity of the enantiomorphs.