title: Purification and properties of beta-ureidopropionase from the rat liver.
abstract: beta-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 327,000 +/- 9000 and 323,000 +/- 13,000 respectively, and the subunit molecular mass was 54,000 +/- 600. The pH optimum for enzyme activity was 7.0 and pI was 6.4. The enzyme catalyzed the amidohydrolysis of N-carbamoyl-beta-alanine and N-carbamoyl-DL-beta-aminoisobutyrate but did not catalyze that of other ureido compounds including N-carbamoyl-DL-aspartate. With N-carbamoyl-beta-alanine and N-carbamoyl-DL-beta-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 microM. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed a sharp break at 19 degrees C.
authors: Faculty of Nutrition, Kobe-Gakuin University, Japan.
authors: N
authors: N
authors: Tamaki
authors: N
authors: N
authors: Mizutani
authors: M
authors: M
authors: Kikugawa
authors: S
authors: S
authors: Fujimoto
authors: C
authors: C
authors: Mizota
mesh_terms: D000494:Allosteric Regulation; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000645:Ammonium Sulfate; D000818:Animals; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000581:Amidohydrolases; C021590:beta-ureidopropionase; D000645:Ammonium Sulfate