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 of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum 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 EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a Km for p-nitrophenylphosphate and fructose-6-phosphate of 370 microM and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and beta-glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.
Wannet
W J
WJ
Department of Microbiology, Faculty of Science, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands.
Wassenaar
R W
RW
Jorissen
H J
HJ
van der Drift
C
C
Op den Camp
H J
HJ
D000135:Acid Phosphatase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000364:Agaricus / Q000201:enzymology*; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005636:Fructosephosphates / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000378:metabolism; D005994:Glycerophosphates / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007526:Isoelectric Point; D008355:Mannitol Phosphates / Q000378:metabolism; D008970:Molecular Weight; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry; D011233:Precipitin Tests; D021122:Protein Subunits / Q000737:chemistry; D013379:Substrate Specificity; D013644:Tartrates / Q000494:pharmacology; D013696:Temperature
D004791:Enzyme Inhibitors; D005636:Fructosephosphates; D005656:Fungal Proteins; D005994:Glycerophosphates; D006023:Glycoproteins; D008355:Mannitol Phosphates; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010710:Phosphates; D021122:Protein Subunits; D013644:Tartrates; C030070:mannitol-1-phosphate; C008644:nitrophenylphosphate; D011092:Polyethylene Glycols; D000249:Adenosine Monophosphate; D019298:Glucose-6-Phosphate; C027618:fructose-6-phosphate; D004492:Edetic Acid; D000135:Acid Phosphatase; C029768:tartaric acid; C031463:beta-glycerophosphoric acid