FIGURE 2.

Activity of Sulf1 and Sulf1 mutants toward heparan sulfate and 4MUS. [³H]Glucosamine-labeled HS material, isolated from Sulf1/2 double knock-out mouse embryonic fibroblasts, was used as a substrate for in vitro activity assays from either cell lysates (A) or conditioned medium (B). The HS substrate was incubated with Sulf1 CA (C87A/C88A), full-length Sulf1, Sulf1ΔHD, -ΔHDB, or -ΔHDC, digested to disaccharides, resolved by strong anion exchange HPLC, and quantified as described under “Experimental Procedures.” Alternatively, activities toward the pseudosubstrate 4-MUS were analyzed from cell lysate samples (C). Due to differences in expression levels (compare Fig. 1B), data were normalized according to band intensities on Western blots or, in the case of untransfected cells, to total protein amounts. Activity of full-length Sulf1 was set to 100%. The error bar at the very left represents the background from endogenous sulfatases in untransfected HT1080 cells. All error bars indicate S.D. from three independent experiments.