Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods.
Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 ( PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST- PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.[1]References
- Monitoring of microcystin-protein phosphatase adduct formation with immunochemical methods. Liu, B.H., Yu, F.Y., Huang, X., Chu, F.S. Toxicon (2000) [Pubmed]
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