Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis o f mannitol in Agaricus bisporus, was purified to homogeneity and characteri zed. The native enzyme appeared to be a high molecular weight type glycopro tein. It has a molecular weight of 145 kDa and consists of four identical 3 9-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maxim um activity occurred at 65 degrees C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDT A, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K-m for p-nitrophenylphosphate and fructose-6-phosphate of 370 mu M and 3. 1 mM, respectively. A broad substrate specificity was observed with signifi cant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-p hosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were deph osphorylated. Antibodies raised against the purified enzyme could precipita te AP activity from a cell-free extract in an anticatalytic immunoprecipita tion test.