Use of a colorimetric protein phosphatase inhibition assay and enzyme linked immunosorbent assay for the study of microcystins and nodularins. Toxicon 32, 1495Toxicon 32, -1507Toxicon 32, , 1994..-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-reactivity with 18 microcystin and nodularin variants was tested. A hydrophobic amino acid, 3-amino-9-methoxy-l0-phenyl-2,6,8-trimethyl-deca-0(E),6(E)-dienoic acid (Adds), which has the (E) form at the C-6 double bond in both microcystin and nodularin, was found essential for these toxins to express antibody specificity. Modification of -000H in glutamic acid of microcystin and nodularin did not alter their antigenicity. Antibody cross-reactivity of these toxins was compared with their ability to inhibit protein phosphatase type 1 (PPI). Detection of PP1 inhibition was done by measuring the inhibition effect of the toxins on p-nitrophenol phosphate activity toward PPI . PP1 was obtained as recombinant PP 1 expressed in E. coli. The inhibition effect of five microcystins and two nodularins on recombinant PP1 activity toward p-nitrophenol phosphate was measured in a microwell plate reader . The concentration of microcystin-LR causing 50% inhibition of recombinant PP1 activity (Ic5 ,) was about 0.3 nM, while that of two modified microcystins had a significantly higher ic,o . Microcystin-LR and nodularin with the (z) form of Adda at the C-6 double bond or having the monoester of glutamic acid did not inhibit PP1 . These three toxins were also nontoxic in the mouse bioassay. These results show the importance of Adda and glutamic acid in toxicity of these cyclic peptides and that PPI inhibition is related to the toxins' mechanism of action .