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 the cytotoxic effects by inhibiting the catalytic activities of phosphatase-2A (PP-2A) and phosphatase-1 (PP-1) and thus disrupt the normal signal transduction pathways.  Microcystins interact with PP-2A and PP-1 by a two-step mechanism involving rapid binding and inactivation of protein phosphatase catalytic subunit, followed by a slower covalent interaction.  It was proposed that inactivation of PP-2A/PP-1 catalytic activity by microcystins precedes covalent adduct formation.  In this study, we used a biosensor based on surface plasmon resonance (SPR) to examine the effects of three microcystins, MCLR, MCRR and MCYR, on the binding between PP-2A and its substrate, phosphorylase-a (PL-a), during the first step of the interaction.  The SPR biosensor provides real-time information on the association and dissociation kinetics of PL-a with immobilized PP-2A in the absence and presence of microcystins.  It was found that the affinity of PL-a to microcystin-bound PP-2A was four times smaller compared to unbound PP-2A, due to 50% decreases in the association rates and two-fold increases in dissociation rates of PL-a binding to PP-2A. The results suggest that the rapid binding of microcystins to the PP-2A catalytic site leads to the formation of a noncovalent microcystin/PP-2A adduct.  While the adduct formation fully inhibits the catalytic activity of PP-2A, it only results in partial inhibition of the substrate binding.  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 biosensor technology in environmental toxicological research.