Microcystins protein phosphatases inhibition

It is obvious from the provisional risk assessment values for microcystins, and, being of the same order of magnitude of mammalian toxicity, similar values may be calculated for the cyanobacterial neurotoxins, that sensitive detection methods are required to detect these low concentrations of toxins. Of the biological methods of detection discussed earlier, the mouse and invertebrate bioassays are not sensitive enough without concentration of water samples, in that they are only able to detect mg of microcystins per litre. Only the immunoassays (ng-/rg 1 and the protein phosphatase inhibition assays (ng O... 

In order to demonstrate the viability of the approach, protein phosphatase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. Two microcystin variants, microcystin-LR and microcystin-RR, were used. Both enzymes were inhibited by these toxins, although to a different extent. The 50% inhibition coefficients (IC50) towards microcystin-LR were 0.50 and 1.40 pgL 1 (concentrations in the microtitre well) for the Upstate and the GTP enzymes, respectively. Hence, the Upstate enzyme was more sensitive. The IC50 towards microcystin-RR were 0.95 and 2.15 pgL-1 for the Upstate and the GTP enzymes, respectively. As expected, microcystin-LR was demonstrated to be a more potent inhibitor. 

J.S. An and W.W. Carmichael, Use of a colorimetric protein phosphatase inhibition assay and enzyme-linked immunosorbent assay for the study of microcystins and nodularins, Toxicon, 32 (1994) 1495-1507. 

C.J. Ward, K.A. Beattie, E.Y.C. Lee and G.A. Codd, Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria comparisons with high-performance liquid chromatography analysis for microcystins, FEMS Microbiol. Lett., 153 (1997) 465-473. 

Protein phosphatase inhibition-based biosensor for amperometric microcystin detection in cyanobacterial cells... 

Carmichael, W.W, and An, J. 1999. Using an enzyme linked immunosorbent assay (ELISA) and a protein phosphatase inhibition assay for the detection of microcystins and nodularins. Nat Toxins 7 377-385. 

Metcalf, J.S., Bell, S.G., and Codd, G.A. 2001. Colorimetric immuno-protein phosphatase inhibition assay for specific detection of microcystins and nodularins of cyanobacteria. Appl Environ Microbiol 67 904—909. 

Rapala, I, Erkomaa, K., Kukkonen, I, Sivonen, K., and Lahti K. 2002. Detection of microcystins with protein phosphatase inhibition assay, high performance liquid chromatography-UV detection and enzymelinked immunosorbent assay. Comparison of methods. Analytica ChimicaActa (466) 213- 231. 

Protein phosphatase inhibition has been correlated with the onset of acute microcystin-induced hepatotoxicosis in mice (70) and with microfilament reorganization and cell deformation in isolated hepatocytes (65). Inhibition of these enzymes causes hyperphosphorylation of numerous cytosolic and cytoskeletal proteins in isolated hepatocytes exposed to microcystin (63,65,71). It has recently been shown that at higher concentrations similar morphological effects are produced also in non-hepatocytes (72). 

Jones, G.J. and Orr, P.T., Release and degradation of microcystin following algicide treatment of a Microcystis aeruginosa bloom in a recreational lake, as determined by HPLC and protein phosphatase inhibition assay. Water Res., 28, 871, 1994. 

Another variant of PP2A assay is the one reported by Isobe et al. [166] where a firefly bioluminescence system is used for the detection of protein phosphatase 2A inhibitors, in which luciferin phosphate is hydrolyzed to luciferin and inorganic phosphate by protein phosphatase 2A. The recent commercial availability of the phosphatase enzymes, which obviates the need to isolate them from animal tissues, also makes this approach very attractive. However, not all microcystins variants react with protein phosphatase enzymes to a similar extent [161,163] and the assay is sensitive to protein phosphatase inhibitors other than microcystins, such as okadaic acid, tautomycin, and calyculin A. In addition, the cyanobacterial sample itself may contain phosphatase activity that masks the presence of toxins [160]. As a consequence, the lack of specificity of the protein phosphatase inhibition assays requires that additional confirmatory analytical methods be employed for specific analysis of cyanobacterial toxins. 

The ELISA is currently the most promising method for rapid sample screening for MCs because of its sensitivity, specificity, and ease of operation. These assays are based on the use of monoclonal or polyclonal antibodies. These assays show greater specificity than protein phosphatase inhibition assays but do not indicate the relative toxicides of microcystin and nodularin variants instead, ELISAs rely on the structure of toxins for detection. Therefore cross-reactivities of the different toxins may vary and sensitivity depends on the structure rather than toxicity. 

C. Mackintosh, Determination of Microcystins in Waters and extracts of cyanobacteria by protein phosphatase inhibition assay, Report to the Department of the Environment (1995). 

Protein phosphatases are ubiquitous. They are foxmd in all tissues and across species as diverse as mammals, plants, and bacteria, and they play a critical role in the regulation of multiple cellular metabolic pathways. Protein phosphatases reverse tiie active state of kinases through the hydrolytic removal of tiie phosphoryl group from kinases. The protein phosphatases inhibited by microcystins have broad substrate specificity and play roles in the regulation of a wide range of cellular fxmc-tions. Protein phosphatase 2A is highly conserved and is a major downregulator of active protein kinases in eukaryotic cells. Toxic effects in hepatocytes and other... 

Useful serine/threonine protein phosphatase inhibitors include microcystin-LR (which inhibits protein phosphatases 1, 2A, and 2C, and related enzymes) and /1-glycerophosphate. Sodium fluoride may also be employed. Sodium orthovanadate inhibits protein tyrosine phosphatases. 

Hepatotoxins include microcystins, which are cyclic heptapeptides (Fig. 5.1a) and cylindrospermopsin, a sulfated guanidinium alkaloid (Fig. 5. lb). Microcystins bind to certain protein phosphatases responsible for regulating the distribution of cytoskeletal proteins (Zurawell et al. 2005 Leflaive and Ten-Hage 2007). Hepatocytes exposed to microcystins eventually undergo cellular deformation, resulting in intra-hepatic bleeding and, ultimately, death (Carmichael 2001 Batista et al. 2003). In contrast, cylindrospermopsin appears to have a different mode of activity, possibly involving inhibition of protein or nucleotide synthesis (Codd et al. 1999 Froscio et al. 2003 Reisner et al. 2004). Nevertheless, microcystins are the most common cyanotoxins isolated from cyanobacterial blooms (Sivonen and Jones 1999). 

Our research group is working on the development of electrochemical biosensors for the detection of microcystin and anatoxin-a(s), based on the inhibition of protein phosphatase and acetylcholinesterase, respectively. These enzyme biosensors represent useful bioanalytical tools, suitable to be used as screening techniques for the preliminary yes/no detection of the toxicity of a sample. Additionally, due to the versatility of the electrochemical approach, the strategy can be applied to the detection of other cyanobacterial toxins. 

On the one hand, protein phosphatase and acetylcholinesterase inhibition assays for microcystin and anatoxin-a(s) detection, respectively, are excellent methods for toxin analysis because of the low limits of detection that can be achieved. On the other hand, electrochemical techniques are characterised by the inherent high sensitivities. Moreover, the cost effectiveness and portability of the electrochemical devices make attractive their use in in situ analysis. The combination of enzyme inhibition and electrochemistry results in amperometric biosensors, promising as biotools for routine analysis. 

Fig. 7.2 Tlie crystal structure of mammalian Ser/Thr protein phosphatase-1, complexed with the toxin mycrocystin was determined at 2.1 A resolution. PPl has a single domain with a fold, distinct from that of the protein tyrosine phosphatases. The Ser/Thr protein phosphatase-1, is a metalloenzyme with two metal ions positioned at the active site with the help of a p-a-p-o-p scaffold. A dinuclear ion centre consisting of Mn2+ And Fe2+ g situated at the catalytic site that binds the phosphate moiety of the substrate. Ser/Thr phosphatases, PPl and PP2A, are inhibited by the membrane-permeable ocadaic acid and by cyclic hexapeptides, known as microcystins. The toxin molecule is depicted as a ball-and-stick structure. On the left and on the ri t, two different views of the same molecule are shown. Microcystin binds to three distinct regions of the phosphatase to the metaLbinding site, to a hydrophobic groove, and to the edge of a C-terminal groove in the vicinity of the active site. At the surface are binding sites for substrates and inhibitors. These ribbon models are reproduced vnth permission of the authors and Nature from ref. 9.

Assays based on the ability of these toxins to inhibit protein phosphatases are a good example of this and are particularly usefirl since they can provide an indication of the biochemical activity of these toxins. These assays are rapid and sensitive and involve the measuring of the inhibitory effect of microcystins on the release of phosphate from phosphoiylated protein substrates (Bell 1994). 

A combination of PPIA and microcystin immunoassay was proposed by Carmichael et al. (1999) to indicate the potential toxicity of a bloom sample and the concentration of the microcystins. A combined assay, consistent with this principle, was developed by Metcalf et al. (2001) this includes preexposure of the sample to microcystin antibodies, to make microcystins/nodularins that are present biounavailable to the subsequent addition of protein phosphatase enzyme, before assaying for protein phosphatase inhibitoiy activity. The resulting assay, termed the colorimetric immunoprotein phosphatase inhibition ass (CIPPIA), was found to be specific for microcystins and nodularins since the microcystin antibodies protect the protein phosphatase from inhibition by the toxins. Complete protection from inhibition of protein phospliatase by the antibodies indicates that the inhibition of the protein phosphatase in the sample was due to the cyanobacterial toxins. These colorimetric assays showed a good correlation with the HPLC analysis of extracts cyanobacteria. Immunoassays can also be combined with physicochemical methods such as HPLC (Zeck 2001b). In this case, the HPLC method separates the microcystins according to their hydrophobicity and the resulting fractions are analyzed by immunoassay. 

Robillot, C., and Hetmion, M.C. 2004. Issues arising when interpreting the results of the protein phosphatase 2A inhibition assay for the monitoring of microcystins. Analytica ChimicaActa 512 339—346. 

Runnegar, M.T., Bemdt, N., Kaplowitz, N. (1995). Microcystin uptake and inhibition of protein phosphatases effects of chemoprotectants and self-inhibition in relation to known hepatic transporters. Toxicol. Appl. Pharmacol. 134 264-72. 

Microcystins have caused the poisoning of wild and domestic animals worldwide, and in 1996, they caused the death of 76 people in Caruaru, Brazil, which was attributed to the use of microcystin-contaminated hemodialysis water. Microcystins, like the well-documented tumor promoter, okadaic acid, strongly and specifically inhibit the protein phosphatases 1 and 2A and have a tumor-promoting activity in the rat liver. In addition to acute hepatotoxicity, microcystins pose problems to human health—which could result from low-level, chronic exposure to microcystins in drinking water, as suggested by the high incidence of primary liver cancer in the... 

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