Purification and Characterization of a Chymotrypsin‐Like Enzyme from Sperm of the Sea Urchin, Hemicentrotus puleherrimus

Abstract

A chymotrypsin-like enzyme was purified from sperm of the sea urchin, H. pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogenous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated MW being 18,500-19,000. This enzyme hydrolyzes N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyze N-benzoyl-L-arginine ethyl ester, N-.alpha.-toluenesulfonyl-L-arginine methyl ester, N-.alpha.-benzoyl-DL-arginine-p-nitroanilide, hippuryl-L-arginine or hippuryl-L-phenylalanine. The Kd for AcTyrOEt and BzTyrOEt are 0.05 mM and 0.0106 mM, respectively. The enzyme activity is inhibited completely by phenylmethylsulfonyl fluoride (PhMeSO2F), chymostatin and L-1-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCH2Cl), and partially by soybean trypsin inhibitor and N-.alpha.-p-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl). The enzyme is activated by CaCl2, MgCl2, NaCl and KCl and loses its activity in 5 min at 67.degree. C. It digests the jelly coat and vitelline layer, not the fertilization membrane. The microvilli of unfertilized eggs elongate and decrease in number as the vitelline layer lyses. The vitelline layer lytic activity is inhibited completely by PhMeSO2F, TosPheCH2Cl and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2Cl and .alpha.1-antitrypsin. Transmission electron microscopy confirmed that this chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported.