title: A protein phosphatase 2A (PP2A) inhibition assay using a recombinant enzyme for rapid detection of microcystins.
abstract: Worldwide blooms of toxic cyanobacteria (blue-green algae) commonly occur in freshwater, often in drinking water sources, necessitating routine monitoring of water quality. Microcystin-LR and related cyanobacterial toxins strongly inhibit protein phosphatase 2A (PP2A) and are therefore assayable by measuring the extent of PP2A inhibition. In this study, we evaluated the suitability of the catalytic subunit of recombinant PP2A (rPP2Ac) expressed with a baculovirus system for use in a microplate microcystin assay. Five microcystin analogs, microcystin-LR, -RR, -YR, -LF, and -LW, and nodularin strongly inhibited rPP2Ac activity with IC(50) values of 0.048, 0.072, 0.147, 0.096, 0.114, and 0.54 nM, respectively. Microcystin-LR in a water sample could be assayed from 0.005 to 5 ng/ml. The assay could detect the toxin at a far lower level than required by the World Health Organization for regulation of microcystin-LR or its equivalent (1 microg/L). Pretreatment or concentration of water samples with low toxin concentrations was not necessary. The microplate assay using rPP2Ac was more sensitive than an enzyme-linked immunosorbent assay (ELISA) method and a cytotoxicity assay. The genetically engineered rPP2Ac was more stable than a commercially available dimeric enzyme, producing accurate and reproducible results. Our results confirm that the rPP2Ac we prepared is an excellent tool for detecting and quantifying microcystins in water.
authors.affiliation: Tropical Technology Center Ltd., 5-1 Suzaki, Uruma, Okinawa, Japan. tikehara@ttc.co.jp
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Ikehara
authors.forename: Shihoko
authors.initials: S
authors.lastname: Imamura
authors.forename: Naomasa
authors.initials: N
authors.lastname: Oshiro
authors.forename: Satsuki
authors.initials: S
authors.lastname: Ikehara
authors.forename: Fukiko
authors.initials: F
authors.lastname: Shinjo
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yasumoto
mesh_terms: D000458:Cyanobacteria / Q000737:chemistry; D004784:Environmental Monitoring / Q000379:methods*; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D005618:Fresh Water / Q000737:chemistry; D052998:Microcystins / Q000032:analysis*; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
chemical_list: D052998:Microcystins; D011993:Recombinant Fusion Proteins; D054648:Protein Phosphatase 2