Figure 9
SDS–PAGE analyses of the main proteases in ESPs capable of hydrolysing collagen I (COI) and fibronectin (Fib). Lane M: protein marker. A ESPs (8.25 μg) hydrolysed COI (4.5 µg) at different pH values. Lane 1: ESPs; Lanes 2, 4, 6 and 8: COI at pH 4.5, 5.5, 6.5 and 7.5, respectively; Lanes 3, 5, 7 and 9: ESPs + COI at pH 4.5, 5.5, 6.5 and 7.5, respectively. B Various ESP doses degraded COI (4.5 µg) at pH 5.5. Lane 1: ESPs; Lane 2: COI; Lanes 3–7: ESPs (5.50, 8.25, 11.00, 13.75 and 16.50 μg, respectively) + COI. C Various inhibitors (1 mM PMSF, 10 mM EDTA, 20 μM E-64 or 10 μM pepstatin) suppressed the ESP-mediated hydrolysis of COI. Lane 1: COI; Lane 2: ESPs + COI; Lane 3: inactivated ESPs + COI; Lane 4: (ESPs + E-64) + COI; Lane 5: (ESPs + PMSF) + COI; Lane 6: (ESPs + pepstatin) + COI; Lane 7: (ESPs + EDTA) + COI. D Inhibitor mixtures suppressed the ESP-mediated hydrolysis of COI. Lane 1: COI; Lane 2: ESPs; Lane 3: ESPs + COI; Lane 4: inactivated ESPs + COI; Lane 5: (ESPs + E-64 + PMSF + pepstatin) + COI; Lane 6: (ESPs + E-64 + PMSF + EDTA) + COI; Lane 7: (ESPs + E-64 + pepstatin + EDTA) + COI; Lane 8: (ESPs + PMSF + pepstatin + EDTA) + COI; Lane 9: (ESPs + E-64 + PMSF + pepstatin + EDTA) + COI; Lane 10: (ESPs + inhibitor cocktail) + COI. E ESPs (8.25 μg) hydrolysed Fib (5 μg) at various pH values. Lane 1: ESPs; Lanes 2, 4, 6 and 8: Fib; Lanes 3, 5, 7, and 9: ESPs + Fib at pH 4.5, 5.5, 6.5 and 7.5, respectively. F Various ESP doses hydrolysed Fib. Lane 1: ESPs; Lane 2: Fib; Lanes 3–8: ESPs (2.5, 5.0, 7.5, 10.0, 12.5 and 15.0 μg, respectively) + Fib. G Inhibitors suppressed the ESP-mediated hydrolysis of Fib. Lane 1: Fib; Lane 2: ESPs + Fib; Lane 3: inactivated ESPs + Fib; Lane 4: (ESPs + E-64) + Fib; Lane 5: (ESPs + PMSF) + Fib; Lane 6: (ESPs + pepstatin) + Fib; Lane 7: (ESPs + EDTA) + Fib. H Inhibitor mixtures suppressed the ESP-mediated hydrolysis of Fib. Lane 1: Fib; Lane 2: ESPs; Lane 3: ESPs + Fib; Lane 4: inactivated ESPs + Fib; Lane 5: (ESPs + E-64 + PMSF + pepstatin) + Fib; Lane 6: (ESPs + E-64 + PMSF + EDTA) + Fib; Lane 7: (ESPs + E-64 + pepstatin + EDTA) + Fib; Lane 8: (ESPs + PMSF + pepstatin + EDTA) + Fib; Lane 9: (ESPs + E-64 + PMSF + pepstatin + EDTA) + Fib; Lane 10: (ESPs + inhibitor cocktail) + Fib.