(A) Inhibition of MLCP by okadaic acid (OA). Enzyme assays in the presence of increasing levels of OA were performed with the indicated substrates at the indicated concentrations; in all assays, the concentration of MLCP was 0.1 nM. Values are presented as a percentage of the average of four control reactions for each substrate without OA. For pCPI-17, each point represents the average, and error bars the standard deviation, of five trials (n = 5); for phosphorylated myosin regulatory light chain (pMRLC), n = 4; for pC-ERMAD, n = 2; for pMyelin Basic Protein (pMyBP), n = 1. Because the MLCP concentration is very low, the IC50s of the pMRLC, pMyBP, and pC-ERMAD reactions must be close to the Ki for OA, which is therefore ~20 nM. The ~10–20× larger IC50 with pCPI-17 indicates that this substrate can compete effectively with OA because its Km is smaller than the Ki. Indeed, the best-fit Km determined by nonlinear regression of this and a similar experiment using 8.75 nM pCPI-17 (n = 3; not shown) is 0.59 ± 0.05 nM (see Supplementary file 1). (B) Inhibition of MLCP by calyculin A. Enzyme assays in the presence of increasing levels of calyculin A were performed with pCPI-17 or with pC-ERMAD at the concentrations indicated; the concentration of MLCP was 0.1 nM. Values are presented as a percentage of the average of three control reactions for each substrate without OA; three replicates of each data point were assayed (n = 3), and error bars represent the standard deviations. The measured Ki of calyculin A for MLCP (from the dephosphorylation of pC-ERMAD) was 0.17 ± 0.05 nM. As with the OA experiments in part (A), the increased IC50 with pCPI-17 (~30×) reflects competition of this substrate with calyculin; the nonlinear regression best-fit is Km = 0.36 ± 0.10 nM for the pCPI-17 dephosphorylation reaction.