Figure 5

Degradation of sculptin by serine proteases and its thrombin inhibition activity. Sculptin (10 µM) was incubated without or with 1 µM of serine protease (thrombin, plasmin, trypsin or factor Xa) in 50 mM phosphate buffer containing 150 mM NaCl and 0.1% PEG 6000, pH 7.4 for 4, 6, 7 or 18 h at 37 °C. The reaction mixtures (20 µl) were separated by SDS-PAGE. (A) SDS-PAGE (15%) of sculptin hydrolysis by serine proteases after 6 h incubation. (B) Percent thrombin inhibition by sculptin after 6 h incubation with serine proteases (see experimental procedures). (C) SDS-PAGE (15%) of sculptin hydrolysis by serine proteases after 18 h incubation. (D) Percent thrombin inhibition by sculptin after 18 h incubation with serine protease (see experimental procedures). The numbering of (B) and (D) correspond to the numbering of (A) and (C) respectively and sculptin control is represented by CTRL. Sculptin (lane 1); thrombin (lane 2) and thrombin with sculptin (lane 3); plasmin (lane 4) and plasmin with sculptin (lane 5); trypsin (lane 6) and trypsin with sculptin (lane 7); factor Xa (lane 8) and factor Xa with sculptin (lane 9) and protein marker (lane 10; in A). (E) Identification of thrombin cleavage sites in sculptin after 7 h of incubation. (F) Identification of factor Xa cleavage sites in sculptin after 4 h of incubation. Thrombin and Factor Xa cleavage sites in sculptin sequence are given in Figs S1 and S2 respectively. The experimental procedure for (C–E) and (F) was same for (A) and (B) except incubation time and type of serine protease used. The results shown in (B) and (D) correspond to the mean ± standard deviation values acquired in three independent experiments; ns nonsignificant, ***p < 0.001 and *p < 0.05.