title: Identification and characterization of hydrophobic microcystins in Canadian freshwater cyanobacteria.
abstract: Hepatotoxic microcystins produced by cyanobacteria in freshwater lakes represent a significant health hazard to humans and agricultural livestock. Liquid chromatography (LC)-linked protein phosphatase (PPase) bioassay analysis of blooms of Microcystis aeruginosa produced in a Canadian drinking water lake identified several PPase inhibitors with significantly greater hydrophobicity than microcystin-LR, based on their retention time on C18 reverse phase LC columns. Seven PPase inhibitors were purified to homogeneity by bioassay-guided fractionation involving Sephadex LH-20 chromatography and two-step reverse phase at pH 6.5 and 2.0. One of the PPase inhibitors, isolated in a final yield of 1.5 micrograms/g lyophilized cyanobacteria, was identified as microcystin-LL by amino acid analysis and mass spectrometry. A further PPase inhibitor (20 ng/g cyanobacteria) was identified as microcystin-LL but with D-Ala replaced by an unknown amino acid. Four PPase inhibitors (< 20 ng/g cyanobacteria) were characterized by amino acid analysis and identified as microcystin-LV, -LM, -LF and -LZ (where Z represents an unknown hydrophobic amino acid). A further microcystin was also identified (< 10 ng/g cyanobacteria) in which arginine was apparently absent. The biological activity of the seven microcystins as inhibitors of the catalytic subunit of protein phosphatase-1 (PP-1c) was compared with microcystin-LR and motuporin (a hydrophobic analogue of nodularin). All of the compounds inhibited PP-1c with IC50 values of 0.06-0.4 nM, consistent with their identification as microcystins. These findings further demonstrate the applicability of a sensitive PPase bioassay for the identification of variant microcystins in the natural environment.
authors.affiliation: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Craig
authors.forename: T L
authors.initials: TL
authors.lastname: McCready
authors.forename: H A
authors.initials: HA
authors.lastname: Luu
authors.forename: M A
authors.initials: MA
authors.lastname: Smillie
authors.forename: P
authors.initials: P
authors.lastname: Dubord
authors.forename: C F
authors.initials: CF
authors.lastname: Holmes
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002170:Canada; D002853:Chromatography, Liquid; D000458:Cyanobacteria / Q000737:chemistry*; D005068:Eutrophication / Q000502:physiology; D052998:Microcystins; D010456:Peptides, Cyclic / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000032:analysis / Q000037:antagonists & inhibitors; D054645:Protein Phosphatase 1; D013056:Spectrophotometry, Ultraviolet; D014871:Water Microbiology; D014881:Water Supply / Q000032:analysis
chemical_list: D000596:Amino Acids; D052998:Microcystins; D010456:Peptides, Cyclic; C078588:microcystin; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1