To determine the pH optimum of enzymatic activity, purified ARSK (Fig. 3B) was PDE10 Inhibitor site incubated for three h at 37 with ten mM pNCS at a variety of pH values amongst 4 and 6, as indicated. Similar amounts on the inactive ARSK-C/A (CA) mutant, purified below the identical circumstances (see Western blot evaluation in the inset) were assayed in parallel. Mean values of two independent experiments S.D. are shown. B, ARSK activity was inhibited by sulfate and phosphate, as tested inside the concentration range from 0.5?0 mM (at 10 mM pNCS). In two independent experiments, IC50 values of two.9 0.two mM (sulfate) and 2.four 0.2 mM (phosphate) were determined. C, the time dependence of pNCS turnover by precisely the same ARSK preparation (35 ng) was measured for as much as 8 h at 37 and pH four.six. D, for measuring the dose dependence, distinct amounts (0 ?five ng) of ARSK have been incubated with ten mM pNCS for four h at 37 and pH 4.6. E and F, the dependence of pNCS and pNPS turnover by 20 ?0 ng of ARSK around the substrate concentration was analyzed at pH 4.6 and 37 . The outcomes were transformed into double-reciprocal Lineweaver-Burk plots RORĪ³ Inhibitor site employing data points from 0.5?0 mM pNCS (E) and 0.five?0 mM pNPS (F). The kinetic constants extrapolated from these plots are offered within the figure.was 20-fold greater as compared with ARSK-C/A (Fig. 4A). In truth, the background activity inside the ARSK-C/A preparation was at the detection limit and, most possibly, because of other contaminating sulfatases. Characterization of ARSK Arylsulfatase Activity–Next we analyzed the enzymatic properties of ARSK and its activity toward arylsulfate pseudosubstrates. To discriminate ARSKassociated sulfatase activity from that of potentially copurified sulfatases, we measured enzymatic activity of ARSK in comparison with ARSK-C/A prepared according to exactly the same purification protocol (see above). ARSK cleaved the tiny aromatic pseudosubstrates pNCS and pNPS (Fig. four) but not the com-monly used pseudosubstrate 4-methylumbelliferyl sulfate (not shown). The apparent pH optimum for ARSK was found to be at an acidic pH of about 4.6 for the pseudosubstrates pNCS (Fig. 4A) and pNPS (not shown), as a result strongly suggesting a lysosomal localization of ARSK. Under the applied assay circumstances (pH 4.6, 37 , 10 mM pNCS, 35 ng ARSK), substrate turnover was linear with time for about 120 min (Fig. 4C). Calculated activities (initial velocities) showed a direct correlation to the level of ARSK present inside the assay (Fig. 4D). Related to other sulfatases, ARSK activity was inhibited by the presence with the reaction solution sulfate or its analog phosphate (17, 29). For ARSK, a moderate sensitivity withVOLUME 288 ?Quantity 42 ?OCTOBER 18,30024 JOURNAL OF BIOLOGICAL CHEMISTRYArylsulfatase K, a Novel Lysosomal SulfataseIC50 values of two.9 0.two mM (sulfate) and 2.4 0.2 mM (phosphate) was observed (Fig. 4B). Substrate saturation curves for pNCS and pNPS have been determined in the pH optimum applying 20 ?0 ng of enzyme/assay. ARSK showed hyperbolic substrate dependence with saturation observed at 15?0 mM for pNCS and 30 ?40 mM for pNPS (not shown). Km and Vmax values have been determined utilizing Lineweaver-Burk plots. From two independent experiments, we calculated a Km of ten.9 3.3 mM for pNCS and 20.six three.6 mM for pNPS (Fig. four, E and F, among the two experiments shown). The maximum specific activity Vmax was extremely equivalent for each substrates, pNCS (0.84 0.29 units/mg, Fig. 4E) and pNPS (0.93 0.16 units/mg, F). In comparison to most other arylsulfatases, these values are substantially reduce than t.