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.
Liu
B H
BH
Department of Food Microbiology & Toxicology, University of Wisconsin-Madison 53706, USA.
Yu
F Y
FY
Huang
X
X
Chu
F S
FS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D000458:Cyanobacteria / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008387:Marine Toxins; D051379:Mice; D008807:Mice, Inbred BALB C; D052998:Microcystins; D010456:Peptides, Cyclic / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011506:Proteins / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins; D013047:Specific Pathogen-Free Organisms; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D011506:Proteins; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C073882:phosphoprotein phosphatase inhibitor 1; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C057862:cyanoginosin LR; C007638:ethanethiol