Fig. 4. Proteolytic activity of silicatein, present in spicule extract from S. domuncula. Extracts were prepared and separated by electrophoresis. After size separation the gel was incubated overnight at room temperature to identify the zones of proteolytic digestion as outlined under Materials and methods. The migration distance of the cleared zone (24/25 kDa) is characteristic for silicatein (lane a). If the sample from the spicules had been pre-incubated with E-64 (lane b) or with the recombinant sponge cystatin (lane c), no clearance zone could be seen. Form these data we conclude that silicatein, present in the spicule extract, still retains proteolytic activity. M, size markers.