title: Biochemical characterization and inhibitory effects of dinophysistoxin-1, okadaic acid and microcystine 1-r on protein phosphatase 2a purified from the mussel Mytilus chilensis.
abstract: Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.
authors.affiliation: Laboratorio Bioquímica de Membrana, Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Casilla 70005, Correo 7, Santiago, Chile.
authors.forename: M
authors.initials: M
authors.lastname: Rivas
authors.forename: C
authors.initials: C
authors.lastname: García
authors.forename: J L
authors.initials: JL
authors.lastname: Liberona
authors.forename: N
authors.initials: N
authors.lastname: Lagos
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008387:Marine Toxins; D052998:Microcystins; D019319:Okadaic Acid / Q000494:pharmacology* / Q000031:analogs & derivatives; D010456:Peptides, Cyclic / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D054648:Protein Phosphatase 2; D011714:Pyrans / Q000494:pharmacology*
chemical_list: D004791:Enzyme Inhibitors; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D011714:Pyrans; D019319:Okadaic Acid; C051904:dinophysistoxin 1; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; C057862:cyanoginosin LR