title: Partial purification and characterization of polyphenoloxidase from culinary-medicinal Royal Sun mushroom (the Himematsutake), Agaricus brasiliensis S. Wasser et al. (Agaricomycetideae).
abstract: The Royal Sun mushroom, the Himematsutake culinary-medicinal mushroom, Agaricus brasiliensis has several polyphenoloxidase activities in a broad sense. Here we report the partial purification of tyrosinase-type polyphenoloxidase (PPO). PPO is purified from A. brasiliensis without browning using a two-phase partitioning with Triton X-114 and ammonium sulfate fractionation. Partially denaturing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide electrophoresis) staining with L-3,4-dihydroxyphenylalanine was performed and the indicated molecular sizes were approximately 70 kDa and 45 kDa. The purified enzyme is in its latent state and can be activated maximally in the presence of 1.6 mM sodium dodecyl sulfate (SDS). This enzyme catalyzes two distinct reactions, monophenolase and diphenolase activity, and the monophenolase activity showed a lag time typical of polyphenoloxidase. The K(m) value for 4-tert-butylcatechol was quite similar in the presence and absence of SDS, but the apparent V(max) value was increased 2.0-fold by SDS. Mimosine was a typical competitive inhibitor with K(i) values of 138.2 microM and 281.0 microM n the presence and absence of SDS, respectively.
authors.affiliation: School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Matsumoto-Akanuma
authors.forename: Satoshi
authors.initials: S
authors.lastname: Akanuma
authors.forename: Masuro
authors.initials: M
authors.lastname: Motoi
authors.forename: Akihiko
authors.initials: A
authors.lastname: Yamagishi
authors.forename: Naohito
authors.initials: N
authors.lastname: Ohno
mesh_terms: D000364:Agaricus / Q000201:enzymology*; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D048690:Fruiting Bodies, Fungal / Q000201:enzymology*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000302:isolation & purification / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D012967:Sodium Dodecyl Sulfate; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase