A fluorescent enzyme inhibition assay for microcystin-LR was developed using a new fluorescent substrate of protein phosphatases 1 (PP1) and 2A (PP2A), 6,8-difluoro-4-methylumbelliferyl phosphate. The PP1 and PP2A inhibition assay for microcystin-LR was performed in a microtiter plate and the fluorescence yielded by the enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The concentration of microcystin-LR causing 50% inhibition of PP1 and PP2A activity (IC50) was 0.01 nM for PP1 and 0.08 nM for PP2A. The measurable range of microcystin-LR was 800 to 0.08 pg/well for both enzymes. The described assay is fast and very sensitive for the detection of microcystin-LR. Furthermore, this assay can be successfully applied to the study of toxins that inhibit PP1 or PP2A.
Copyright 1999 Academic Press.