title: A novel alkaline protease with antiproliferative activity from fresh fruiting bodies of the toxic wild mushroom Amanita farinosa.
abstract: A novel protease with a molecular mass of 15 kDa was purified from fresh fruiting bodies of the wild mushroom Amanita farinosa. The purification protocol entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and SP-Sepharose. It demonstrated a single 15-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and a 15-kDa peak in gel filtration. The optimal pH and optimal temperature of the protease were pH 8.0 and 65 °C, respectively. Proliferation of human hepatoma HepG2 cells was inhibited by the protease with an IC(50) of 25 µM. The protease did not have antifungal or ribonuclease activity.
authors.affiliation: State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Sun
authors.forename: Yongchang
authors.initials: Y
authors.lastname: Zhao
authors.forename: Hongmei
authors.initials: H
authors.lastname: Chai
authors.forename: Hexiang
authors.initials: H
authors.lastname: Wang
authors.forename: Tzi Bun
authors.initials: TB
authors.lastname: Ng
mesh_terms: D000327:Adsorption; D000545:Amanita / Q000201:enzymology*; D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D057075:Enzyme Assays; D048690:Fruiting Bodies, Fungal / Q000201:enzymology*; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D056945:Hep G2 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013696:Temperature
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D005656:Fungal Proteins; D010447:Peptide Hydrolases