To figure out the pH optimum of enzymatic activity, purified ARSK (Fig. 3B) was incubated for 3 h at 37 with 10 mM pNCS at several pH values between 4 and 6, as indicated. Comparable Caspase-3/CASP3 Protein Accession amounts of your inactive ARSK-C/A (CA) mutant, purified beneath exactly the same circumstances (see Western blot evaluation within the inset) had been assayed in parallel. Imply values of two independent experiments S.D. are shown. B, ARSK activity was inhibited by sulfate and phosphate, as tested within the concentration range from 0.5?0 mM (at 10 mM pNCS). In two independent experiments, IC50 values of 2.9 0.2 mM (sulfate) and two.four 0.2 mM (phosphate) had been determined. C, the time dependence of pNCS turnover by the exact same ARSK preparation (35 ng) was measured for as much as 8 h at 37 and pH 4.6. D, for measuring the dose dependence, different amounts (0 ?five ng) of ARSK have been incubated with ten mM pNCS for 4 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 results had been transformed into double-reciprocal Lineweaver-Burk plots using data points from 0.five?0 mM pNCS (E) and 0.five?0 mM pNPS (F). The kinetic constants extrapolated from these plots are given within the figure.was 20-fold larger as compared with ARSK-C/A (Fig. 4A). Actually, the background activity within the ARSK-C/A preparation was at the detection limit and, most in all probability, 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 ready according to exactly the same purification protocol (see above). ARSK cleaved the small aromatic pseudosubstrates pNCS and pNPS (Fig. 4) but not the com-monly used pseudosubstrate 4-methylumbelliferyl sulfate (not shown). The apparent pH optimum for ARSK was identified to be at an acidic pH of about 4.six for the pseudosubstrates pNCS (Fig. 4A) and pNPS (not shown), hence strongly suggesting a lysosomal localization of ARSK. Beneath the applied assay conditions (pH 4.six, 37 , ten 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 for the amount of ARSK present in the assay (Fig. 4D). Equivalent to other sulfatases, ARSK activity was inhibited by the presence with the reaction product sulfate or its analog phosphate (17, 29). For ARSK, a Adrenomedullin/ADM Protein Formulation moderate sensitivity withVOLUME 288 ?Number 42 ?OCTOBER 18,30024 JOURNAL OF BIOLOGICAL CHEMISTRYArylsulfatase K, a Novel Lysosomal SulfataseIC50 values of two.9 0.two mM (sulfate) and two.4 0.2 mM (phosphate) was observed (Fig. 4B). Substrate saturation curves for pNCS and pNPS were determined at the pH optimum making use of 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 were determined utilizing Lineweaver-Burk plots. From two independent experiments, we calculated a Km of 10.9 three.3 mM for pNCS and 20.6 3.6 mM for pNPS (Fig. 4, E and F, one of the two experiments shown). The maximum distinct activity Vmax was extremely related 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 significantly lower than t.