title: Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina.
abstract: The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.
authors.affiliation: Department of Life Science and Biotechnology, Shimane University, Matsue, Shimane 690-8504, Japan.
authors.forename: Md Abul Kalam
authors.initials: MA
authors.lastname: Azad
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Sawa
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shibata
mesh_terms: D020134:Catalytic Domain; D035264:Flowers / Q000201:enzymology*; D020035:Holoenzymes / Q000378:metabolism*; D019319:Okadaic Acid / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification*; D054648:Protein Phosphatase 2; D031447:Tulipa / Q000201:enzymology*
chemical_list: D020035:Holoenzymes; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2