Purification and Characterization of the Major Acid Phosphatase Isozyme Secreted by Maize Endosperm Cultures

The major acid phosphatase [EC 3.1.3.2J isozyme secreted by maize endosperm cultures was purified from concentrated culture filtrates by (NH4)2S04 fractionation and chromatography on columns of diethylaminolethylSephadex A-25, concanavalin A-Sepharose, and Sephacryl S200. The native and subunit M, value's for the purified phosphatase were 29000 and 31500, respectively. The purified enzyme contained approximately 6% neutral sugars, primarily mannose, fucose and xylose. Using p-nitrophenyl-phosphate as the substrate, there was maximum in vitro activity at 50 and pH 5.5 K m values for p-nitrophenyl-phosphate, sn-glycerol-3-phosphate, glucose-6-phosphate, and ATP were 0.35 mM, 0.26 mM, 1.2 mM, 6.7 mM, respectively. With p-nitrophenyl-phosphate as the substrate, the maize endosperm acid phosphatase was inhibited by inorganic phosphate, fluoride and molybdate, with K i values of 0.56 mM, 0.40 mM and 1.2 mM, respectively.