Fig. 7. Arylsulfatase activity of expressed Sulfs and lack of the activity in HSulf mutants.

HSulf-1, HSulf-2, or their mutated forms (HSulf-1 ΔCC and HSulf-2 ΔCC) were purified from the conditioned medium of transfected CHO cells by binding to Ni-NTA beads. A, the bead-bound material was tested for arylsulfatase activity as a function of time against 10 mM 4-MUS substrate at pH 8. The “no enzyme” control was based on testing conditioned medium from vector control-transfected CHO cells. No activity was detected in the absence of added substrate (not shown). The same results were obtained in three different experiments. B, the concentrated conditioned medium was tested for arylsulfatase activity at pH 8 for 2 h at different concentrations (1–10 mM) of 4-MUS. To eliminate background effects, the activity in vector control material was subtracted from that of Sulf-transfected material. C, the eluted material from Ni-NTA was tested for arylsulfatase activity at pH 8 for 2 h as a function of input volume of conditioned medium. The same results were obtained in three different experiments. D, bead-bound Sulfs were tested for arylsulfatase activity (1 h) at the indicated pH values. The activity of each Sulf was determined relative to that of beads exposed to an equivalent volume of vector-control conditioned medium. The activity of HG6S was determined (24-h incubation) relative to that of the buffer. The same results were obtained in three different experiments.