Extended Data Figure 8: Substrates and inhibitors of hNOTUM.

a, Inhibition of hNOTUM activity on pNP-butyrate (pNP4) by PMSF (30 min pre-incubation with 2 mM PMSF) as well as by Triton X-100 and CHAPS (0.5%). Presence of 20 mM SOS and 50 mg l⁻¹ heparin results in a minor increase of esterase activity. The height of each bar represents activity relative to the mean of four control samples lacking the additives. b, Saturable inhibition of hNOTUM by Triton X-100. Triton X-100 inhibits many esterases owing to binding to the acyl binding pocket through its hydrophobic group. c, Lack of inhibition of Norrin-mediated β-catenin stabilization by Notum. Recombinant Norrin was pretreated with hNOTUM_core at a concentration sufficient to suppress Wnt3A-mediated signalling. d, e, Saturation kinetics of the action of hNOTUM on pNP-octanoate (pNP8, d) and pNP-butyrate (pNP4, e). The activity was normalized to the A_max calculated for hNOTUM_core. The activity values for the larger, full length protein were adjusted to compensate for the increased mass. Apparent K_m values in d were corrected for the inhibition caused by Triton X-100. f, Saturation inhibition kinetics with myristoleic and palmitoleic acid. pNP8 was used at a concentration of 1 mM and 250 μM, respectively.