title: An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases.
abstract: An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.
authors.affiliation: Department of Anatomy and Cell Biology, University of Bergen, Norway.
authors.forename: M H
authors.initials: MH
authors.lastname: Serres
authors.forename: K E
authors.initials: KE
authors.lastname: Fladmark
authors.forename: S O
authors.initials: SO
authors.lastname: Døskeland
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D001681:Biological Assay; D007455:Iodine / Q000737:chemistry; D008387:Marine Toxins; D052998:Microcystins; D019319:Okadaic Acid / Q000737:chemistry / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000032:analysis / Q000737:chemistry / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012623:Seawater / Q000032:analysis; D012758:Shellfish / Q000032:analysis; D013997:Time Factors; D014020:Tissue Extracts / Q000032:analysis; D014118:Toxins, Biological / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D014881:Water Supply / Q000032:analysis
chemical_list: D001427:Bacterial Toxins; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D014020:Tissue Extracts; D014118:Toxins, Biological; C063998:nodularin; D019319:Okadaic Acid; D007455:Iodine; D010749:Phosphoprotein Phosphatases; C057862:cyanoginosin LR