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

An acid phosphatase (EC 3.1.3.2) has been identified and purified from cast or bean (Ricinus communis L., IAC-80) seed through sulphopropyl (SP)-Sephad ex, diethylaminoethyl (DEAE)-Sephadex, Sephacryl S-200, and Concanavalin A- Sepharose chromatography. The enzyme was purified 2000-fold to homogeneity, with a final specific activity of 3.8 mu kat mg(-1) protein. The purified enzyme revealed a single diffuse band with phosphatase activity on nondenat uring polyacrylamide gel electrophoresis, at pH 8.3. The relative molecular mass, determined by high-performance liquid chromatography (HPLC), was fou nd to be 60 kDa, The acid phosphatase had a pH optimum of 5.5 and an appare nt K-m value for p-nitrophenylphosphate of 0.52 mM, The enzyme-catalyzed re action was inhibited by inorganic phosphate, fluoride, vanadate, molybdate, p-chloromercuribenzoate (pCMB), Cu2+ and Zn2+, The strong inhibition by pC MB, Cu2+ and vanadate suggests the presence of sulfhydryl groups essential for catalysis. The castor bean enzyme also recognized tyrosine-phosphate an d inorganic pyrophosphate (PPi) as substrate. The highest specificity const ant (V-max/K-m) was observed with PPi, making it a potential physiological substrate.