Purification and kinetic properties of a castor bean seed acid phosphatase containing sulfhydryl groups

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 K m 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), Cu 2+ and Zn 2+ . The strong inhibition by p CMB, Cu 2+ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine‐phosphate and inorganic pyrophosphate (KPP i ) as substrate. The highest specificity constant (V max /K m ) was observed with KPP i , making it a potential physiological substrate.