Purification and Characterization of Creatinine Amidohydrolase of Alcaligenes Origin

概要

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Creatinine amidohydrolase (creatininase) from Alicaligenes sp. nov. was purified to electrophoretic homogeneity by adsorption on diethylaminoethyl-cellulose, affinity chromatography on creatinyl-AH-Sepharose, gel filtration on Sephadex G-200 and hydroxyapatite chromatography. The molcular weight of the enzyme was estimated to be approximately 160000 by gel filtration on Sephadex G-200 and 80000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the enzyme was assumed to consist of two identical subunits. The enzyme showed maximum activity at pH 7-8 and was stable in the pH range of 8-11.5. The enzyme catalyzed interconversion between creatinine and creatine, and the K_m values for creatinine and creatine were 60.9 mM and 162 mM, respectively. Though the enzyme was markedly inactivated by ethylenediamine-tetraacetate (EDTA), N-bromosuccinimide, Zn^<2+>, Cu^<2+>, Ni^<2+> or Co^<2+>, activation of the enzyme was only observed in the presence of Mn^<2+>. Furthermore, the reactivation of EDTA-treated inactive enzyme was observed on the addition of Mn^<2+> to the reaction mixture.
社団法人日本薬学会の論文
1986-01-25