Figure 5

Characterization of inhibitor binding to the active site of WT-SPD and MI-SPD. (A) WT- or (B) MI-SPD (2 μg/ml) was captured on rabbit anti-FSAP polyclonal antibody-coated wells (5 μg/ml). Biot-PPACK (●, 0–2 μM), Biot-aprotinin (■, 0–2 μM) or Biot-Mab570 (▲, 0–2 μg/ml) at different concentrations was added as indicated and specific binding was determined using streptavidin POD as absorbance at 450 nm (OD, mean + SD). (C) In a similar experiment as in (A,B) Biot-PPACK (0.1 μM) binding to WT-SPD (●, ■) and MI-SPD (▲, ▼) was tested in the presence or absence of increasing concentrations of PPACK (●, ▲, 0–1 μM) or aprotinin (■, ▼, 0–1 μM). (D) The proteolytic activity of WT-SPD against S-2288 was determined in the presence of increasing concentrations of Biot-PPACK (▲) and PPACK (∗, μM), Biot-aprotinin (■) and aprotinin (◆, μM), Biot-Mab570 (●) and Mab570 (▼, μg/ml). Enzyme activity is presented as mOD/min (mean + SD). All binding and activity studies were done in the presence of 2 mM Ca²⁺ and 137 mM Na⁺.