Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups |
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Authors: | Paulo Afonso Granjeiroa Carmen Veríssima Ferreirab José Mauro Granjeiroc Eulázio Mikio Tagac Hiroshi Aoyamaa |
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Institution: | Departamento de Bioquimica, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970 Campinas, São Paulo, Brasil; Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, 59078-000 Natal, Rio Grande do Norte, Brasil; Departamento de Ciências Biológicas, Faculdade de Odontologia de Bauru, Universidade de São Paulo, 17043-101 Bauru, São Paulo, Brasil |
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Abstract: | An acid phosphatase (EC 3.1.3.2) has been identified and purified from castor bean ( Ricinus communis L., IAC-80 ) seed through sulphopropyl (SP)-Sephadex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A-Sepharose chromatography. The enzyme was purified 2 000-fold to homogeneity, with a final specific activity of 3.8 μkat mg−1 protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenaturing polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was found to be 60 kDa. The acid phosphatase had a pH optimum of 5.5 and an akpparent Km value for p -nitrophenylphosphate of 0.52 m M . The enzyme-catalyzed reaction was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p -chloromercuribenzoate ( p CMB), Cu2+ and Zn2+. The strong inhibition by p CMB, Cu2+ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate and inorganic pyrophosphate (KPPi) as substrate. The highest specificity constant (Vmax/Km) was observed with KPPi, making it a potential physiological substrate. |
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