Purification and Characterization of Trypsin like Enzyme of Sperm of the Sea Urchin, Hemicentrotus pulcherrimus: (sea urchin/sperm/trypsin like enzyme/proteolytic enzyme).

Abstract

Trypsin like enzyme has been isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus, using tryptophane methyl ester-Sepharose 4B and soybean trypsin inhibitor-Sepharose 4B affinity chromatographies. The isolated enzyme preparation is homogenous in polyacrylamide gel electrohoresis at pH 2.3. The molecular weight of the enzyme estimated by gel filtration is about 33,000, and the enzyme separates into two subunits of 10,900 and 20,500 on sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol. This enzyme is active to N-α-benzoyl arginine ethyl ester (BAEE), N-α-toluenesulfonyl-L-arginine ethyl ester (TAME), and N-α-benzoyl-DL-arginine-p-nitroanilide, but not N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-tyrosine ethyl ester, Hippuryl-L-arginine, and Hippuryl-L-phenylalanine. The optimal pH of this enzyme is about 8.0. The Michaelis constants for BAEE and TAME are 3.3 × 10-6 M, and 8.2 × 10-5 M, respectively. Soybean trypsin inhibitor and lima bean trypsin inhibitor completely inhibit the activity of this enzyme, while N-α-tosyl-L-lysine chloromethyl ketone, ovomucoid trypsin inhibitor, and α-1-antitrypsin partially inhibit. L-1-tosylamide-2-phenyl chloromethyl ketone, chyrnostatin, and aporotinine are without effect. This enzyme is stable at pH 2.0-3.0 and labile at pH 8.0. Ca2+ and Mg2+ activate this enzyme, but do not stabilize at pH 8.0. Seawater, NaCl, and KCl inhibit this enzyme activity. Release of this enzyme from the acrosomal vesicle is suggested.