title: Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria: comparisons with high-performance liquid chromatographic analysis for microcystins.
abstract: Microcystins are cyclic heptapeptide hepatotoxins commonly produced by bloom-forming genera of cyanobacteria. These toxins are potent and specific inhibitors of protein phosphatases 1 and 2A. We have optimised a rapid, simple and sensitive colorimetric protein phosphatase 1 inhibition assay, utilising the activity of protein phosphatase 1 as expressed in a recombinant strain of Escherichia coli, towards the chromogenic substrate, p-nitrophenyl phosphate. A standard curve for the inhibition of protein phosphatase 1 by microcystin-LR was constructed with an IC50 of about 38 ng ml-1 and a limit of detection of 10-20 ng ml-1. Twenty-three laboratory-grown strains and 25 natural bloom samples of cyanobacteria were analysed by high-performance liquid chromatography for microcystins and by the protein phosphatase 1 inhibition assay. Agreement for the microcystin contents of the samples detected by high-performance liquid chromatography and the protein phosphatase 1 inhibition assay showed good correlation (R2 > 0.93, P < 0.0001). The suitability of the colorimetric protein phosphatase 1 inhibition assay as a screen for cyanobacterial microcystins is discussed.
authors.affiliation: Department of Biological Sciences, University of Dundee, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Ward
authors.forename: K A
authors.initials: KA
authors.lastname: Beattie
authors.forename: E Y
authors.initials: EY
authors.lastname: Lee
authors.forename: G A
authors.initials: GA
authors.lastname: Codd
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D000458:Cyanobacteria / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D007202:Indicators and Reagents; D000432:Methanol; D052998:Microcystins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010456:Peptides, Cyclic / Q000032:analysis* / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D054645:Protein Phosphatase 1; D012680:Sensitivity and Specificity; D012997:Solvents
chemical_list: D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D052998:Microcystins; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010456:Peptides, Cyclic; D012997:Solvents; C008644:nitrophenylphosphate; C078588:microcystin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D000432:Methanol