INHIBITION OF SPECIFIC BINDING OF OKADAIC ACID TO PROTEIN PHOSPHATASE 2A BY MICROCYSTIN-LR, CALYCULIN-A AND TAUTOMYCIN - METHOD OF ANALYSIS OF INTERACTIONS OF TIGHT-BINDING LIGANDS WITH TARGET PROTEIN

Several groups have reported that okadaic acid (OA) and some other tight-binding protein phosphatase inhibitors including microcystin-LR (MCLR), calyculin-A and tautomycin prevent each other from binding to protein phosphatase 2A (PP2A). In this paper we have introduced an improved procedure for examining to what extent the affinity of an enzyme for a labelled tight-binding ligand is reduced by binding of an unlabelled tight-binding ligand to the enzyme. Using this procedure, we have analysed the dose-dependent reduction of PP2A binding of [24-H-3]OA by addition of OA, MCLR, calyculin-A and tautomycin. The results indicate that the binding of the unlabelled inhibitors to the PP2A molecule causes a dramatic (10(6)-10(8)-fold) increase in the dissociation constant associated with the interaction of [24-H-3]OA and PP2A. This suggests that OA and the other inhibitors bind to PP2A in a mutually exclusive manner. The protein phosphatase inhibitors may share the same binding site on the PP2A molecule. We have also measured values of the dissociation constant (K-i) for the interaction of these toxins with protein phosphatase 1 (PP1). For MCLR and calyculin-A, the ratio of the K-i value obtained for PP1 to that for PP2A was in the range 4-9, whereas it was 0.01-0.02 for tuatomycin. The value of tautomycin is considerably smaller than that (0.4) calculated from previously reported K-i values.