USE OF A COLORIMETRIC PROTEIN PHOSPHATASE INHIBITION ASSAY AND ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE STUDY OF MICROCYSTINS AND NODULARINS

Microcystins and nodularins are cyclic peptide hepatotoxins and tumor promoters produced by several genera of cyanobacteria. Using a rabbit anti-microcystin-LR polyclonal antibody preparation, the cross-reactiv ity with 18 microcystin and nodularin variants was tested. A hydrophob ic amino acid, y-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (Adda), which has the (E) form at the C-6 double bond in both microcy stin and nodularin, was found essential for these toxins to express an tibody specificity. Modification of -COOH in glutamic acid of microcys tin and nodularin did not alter their antigenicity. Antibody cross-rea ctivity of these toxins was compared with their ability to inhibit pro tein phosphatase type 1 (PP1). Detection of PP1 inhibition was done by measuring the inhibition effect of the toxins on p-nitrophenol phosph ate activity toward PP1. PP1 was obtained as recombinant PP1 expressed in E. coli. The inhibition effect of five microcystins and two nodula rins on recombinant PP1 activity toward p-nitrophenol phosphate was me asured in a microwell plate reader. The concentration of microcystin-L R causing 50% inhibition of recombinant PP1 activity (IC50) was about 0.3 nM, while that of two modified microcystins had a significantly hi gher IC50. Microcystin-LR and nodularin with the (z) form of Adda at t he C-6 double bond or having the monoester of glutamic acid did not in hibit PP1. These three toxins were also nontoxic in the mouse bioassay . These results show the importance of Adda and glutamic acid in toxic ity of these cyclic peptides and that PPI inhibition is related to the toxins' mechanism of action.