P-Ureidopropionase was purified 1000-fold over the initial rat liver extract, using heat treatment, ammonium sulfate fractionation, CM-Sepharose CL-6B, DEAE-Sepharose CL-6B, hydroxyapatite and Sephacryl S-300 chromatographies. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. Its molecular mass, determined by gel filtration and sucrose density gradient centrifugation, was 327000 9000 and 323000 5 13000 respectively, and the subunit molecular mass was 54000 _+ 600. The pH optimum for enzyme activity was 7.0 and PI was 6.4. The enzyme catalyzed the amidohydrolysis of N-carbamoyl-P-alanine and N-carbamoyl-DL-P-aminoisobutyrate but did not catalyze that of other ureido compounds including N-carbamoyl-DL-aspartate.With N-carbamoyl-P-alanine and N-carbamoyl-DL-P-aminoisobutyrate as substrate, the enzyme exhibited positive cooperativity with a Hill coefficient h = 2. The enzyme activity was proportional to the enzyme concentration between 0.2 nM and 0.5 pM. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed a sharp break at 19 "C.It is well known that uracil catabolizes to p-alanine via dihydrouracil and N-carbamoyl-P-alanine. /?-Ureidopropionase (N-carbamoyl-P-alanine amidohydrolase) is the last of the uracil-degradative enzymes [l, 21. The key role of uracil metabolism may be to regulate the pyrimidine pool. Canellakis [3] and Fritzson [4, 51 proposed that the first enzyme of the pathway, dihydrouracil dehydrogenase, was rate-limiting for pyrimidine degradation. Under limited conditions, however, P-ureidopropionase becomes ratelimiting [6, 71.During differentiation [8] and fetal and neonatal development 191 there is an increase in the activity of the catabolic pathway of pyrimidine and the activity of P-ureidopropionase increases in parallel with the overall pathway. In contrast the synthetic utilization of uridine into RNA declined sharply with fetal and neonatal development [9]. From these findings, 0-ureidopropionase might have an important function in uracil metabolism. We purified P-ureidopropionase from rat liver and studied its properties. In the course of the studied we ascertained the allosteric properties of the enzyme. MATERIALS AND METHODS MaterialsAll chemicals used were of analytical grade and were purchased from Nakarai Chemicals Ltd (Kyoto) unless otherwise stated. N-Carbamoyl-P-alanine and other carbamoyl Correspondence to N. Tamaki, Laboratory of Nutritional Chemistry, Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe, Japan 673Enzymes. p-Ureidopropionase or N-carbamoyl-P-alanine amidohydrolase (EC 3.5.1.6); dihydrouracil dehydrogenase (EC 1.3.1.2); dihydropyrimidinase (EC 3.5.2.2); RNA nucleotidyltransferase (EC 2.7.7.6); pyruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); alcohol dehydrogenase (EC 1.1.1.1).compounds were products of Sigma Chemicals. CMSepharose CL-6B, DEAE-Sepharose CL-6B and Sephacryl S-300 were obtained from Pharmacia and hydroxy...

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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.

Summary ƒÀ-Alanine-oxoglutarate aminotransferase (ƒÀ-Ala-T

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