Effects of microcystins on phosphorylase-a binding to phosphatase-2A: kinetic analysis by surface plasmon resonance biosensor

Cyclic heptapeptide microcystins are a group of hepatoxicants which exert t he cytotoxic effects by inhibiting the catalytic activities of phosphatase- 2A (PP-2A) and phosphatase-l (PP-1) and thus disrupt the normal signal tran sduction pathways. Microcystins interact with PP-2A and PP-I by a two-step mechanism involving rapid binding and inactivation of protein phosphatase c atalytic subunit, followed by a slower covalent interaction. It was propose d that inactivation of PP-2A/PP-1 catalytic activity by microcystins preced es covalent adduct formation. In this study, we used a biosensor based on s urface plasmon resonance (SPR) to examine the effects of three microcystins , MCLR, MCRR and MCYR, on the binding between PP-2A and its substrate. phos phorylase-a (PL-a), during the first step of the interaction. The SPR biose nsor provides real-time information on the association and dissociation kin etics of PL-a with immobilized PP-2A in the absence and presence of microcy stins. It was found that the affinity of PL-a to microcystin-bound PP-2A wa s four times smaller compared to unbound PP-ZA, due to 50% decreases in the association rates and two-fold increases in dissociation rates of PL-a bin ding to PP-2A. The results suggest that the rapid binding of microcystins t o the PP-ZA catalytic site leads to the formation of a noncovalent microcys tin/PP-2A adduct. While the adduct formation fully inhibits the catalytic a ctivity of PP-2A, it only results in partial inhibition of the substrate bi nding. The similar effects of the three microcystins on PP-2A suggest that the toxins bind to PP-2A at the same site and cause similar conformational changes. The present work also demonstrates the potential application of bi osensor technology in environmental toxicological research. (C) 1999 Elsevi er Science B.V. All rights reserved.