Purification and characterization of a chymotrypsin-like enzyme from sperm of the sea urchin, Hemicentrotus pulcherrimus.
A chymotrypsin-like enzyme has been purified from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophan methyl ester (TrpOMe) linked to Sepharose 4B as an affinity column for chromatography and gel filtration. The isolated enzyme preparation is homogeneous in sodium dodecylsulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 18,500-19,000. This enzyme hydrolyses N-acetyl-L-tyrosine ethyl ester (AcTyrOEt) and N-benzoyl-L-tyrosine ethyl ester (BzTyrOEt); the optimal pH is 8.0. It does not hydrolyse 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 Michaelis constants 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 degrees 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. We have confirmed by transmission electron microscopy that our chymotrypsin-like enzyme completely digests the vitelline layer. A result implying release of this enzyme from the acrosome vesicle is also reported.
D000177:Acrosome / Q000201:enzymology; D000818:Animals; D002918:Chymotrypsin / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D009391:Nephelometry and Turbidimetry; D012617:Sea Urchins / Q000201:enzymology*; D013094:Spermatozoa / Q000201:enzymology*; D014817:Vitelline Membrane / Q000201:enzymology
D002918:Chymotrypsin