FIGURE 3.

Enzyme kinetics of Sulf1ΔHDC with 4-MUS. Secreted Sulf1ΔHDC was enriched from conditioned medium of stably transfected HT1080 cells and analyzed with 4-MUS as a substrate. The activities at different pH values (pH 5–9 at 37 °C) (A) or temperatures (4–65 °C at pH 7.0) (B) were investigated at a substrate concentration of 2 mm 4-MUS using 60 ng of Sulf1ΔHDC and a 1-h incubation time. The enzymatic stability of Sulf1ΔHDC was investigated at pH 7.0 and 37 °C under conditions not limited by substrate concentrations (C). Furthermore, the substrate dependence of the 4-MUS turnover was monitored in the range between 0 and 50 mm 4-MUS at 37 °C and pH 7.0 using 40 ng of Sulf1ΔHDC per reaction. Turnover rates were determined from a calibration curve with 4-MUS. At high concentrations, substrate inhibition was observed (D). The effects of inorganic phosphate (E) and sulfate (F) on the activity of Sulf1ΔHDC were analyzed at a 4-MUS concentration of 10 mm.