Hatching enzyme from the sea-squirt Ciona intestinalis: purification and properties

Abstract

We have purified a 34 kDa hatching enzyme from the water in which the embryos of the sea-squirt Ciona intestinalis hatch. This enzyme was obtained in homogeneous form as judged from SDS-PAGE and HPLC gel filtration. The enzyme possesses proteolytic activity and is able to digest the chorion of the egg of C. intestinalis. It is a metalloproteinase and contains one atom of Zn per molecule. The optimum pH is 8.5. The enzyme shows hydrolytic activity towards the -CO–NH- bonds, which are hydrolyzed by the members of the serine proteinase family. It has a trypsin-like activity in that it cuts the bond of Arg and Lys at P 1 position of the scissile bond -P 1–P 1′, but it differs from trypsin insofar as it hydrolyzes the peptide bond on either side of Arg and Lys. The purified enzyme is inhibited by the common metal-chelators and by the classical trypsin proteinase inhibitors. The apparent K m values at 37°C and pH 8.5 toward tosyl-Gly-Pro-Arg-NHNap, tosyl-Gly-Pro-Lys-NHNap and Bz-Arg-Gly-Arg-NHNap were 0.125, 0.5 and 2.5 mM, respectively. The results obtained in this study suggest that the hatching enzyme from C. intestinalis exhibits both trypsin-like activity and metalloproteinase activity.