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

Abstract

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 homogenous 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-a-toluenesulfonyl-L-arginine methyl ester, N-a-benzoyl-DL-argininep-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 (PhMeS02F), chymostatin and L-I-tosylamido-2-phenylethyl chloromethyl ketone (TosPheCHKl), and partially by soybean trypsin inhibitor and N-a-p-tosyl-L-lysine chloromethyl ketone (TosLysCHzCl). The enzyme is activated by CaC12, MgC12, NaCl and KCl, and loses its activity in 5 min at 67 "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 PhMeSOzF, TosPheCHzCl, and chymostatin, and partially by soybean trypsin inhibitor, TosLysCH2C1, and al-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 Vitelline layer lysins are found in the sperm of a variety of animals such as Megathura [1,2], Tegula [3,4], Hydroides[5], Mytilus [6], Bufo [7] and some mammals [8 -lo]. Although it has long been throught that sea urchin sperm too have a vitelline layer lysin, only a brief report is available [ll]. A trypsin-like enzyme has been purified from sperm of Strongylocentrotuspurpuratus, but it was not shown whether this enzyme has the vitelline layer lytic activity [12]. According to a recent report [13], an arylsulfatase exists in both spermatozoa and seminal plasma of Hemicentrotus pulcherrimus and Strongylocentrotus intermedius and digests the jelly coat and vitelline layer. It is also suggested that a chymotrypsin-like enzyme participates in sperm penetration [14].In the preceding paper [15], we have shown that a chymotrypsin-like enzyme extracted from H. pulcherrimus sperm partially digests the vitelline layer, either intact or isolated, of the same species. Disruption by this enzyme preparation of some protein components of the vitelline layer has also been demonstrated by sodium dodecylsulfate/polyacrylamide gel electrophoresis. In this paper, purification and partial characterization of this enzyme is described. MATERIALS AND METHODS Crude ExtractGametes of the sea urchin Hemicentrotus pulcherrimus were...

Dephosphorylation of a major sperm membrane protein is induced by egg jelly during sea urchin fertilization

“…Intracellular cAMP and cGMP levels also increase in response to jelly (16), and enzymatic activities other than those involved in cyclic nucleotide metabolism, including protease (17,18), protein kinase (19), protein phosphatase (20), and phospholipase (21,22), have been reported to increase or to be released upon jelly treatment. Thus, a number of molecular responses of sperm to treatment with egg jelly have been identified, but the complete sequence of reactions by which jelly activates the spermatozoan remains unknown.…”

Effects of Proteasome Inhibitors on Fertilization of the Sea Urchin Anthocidaris crassispina

“…A 19-kDa chymotrypsin-like protease has been purified from sea urchin sperm. 11) This protease is currently proposed to be a vitelline-coat lysin, since the purified enzyme has a vitellineenvelope lytic activity. However, since the vitelline-coat-lytic activity and the acetyl-tyrosine ethyl ester-hydrolyzing activity of the purified enzyme is completely inhibited by low concentrations (100-200 mM) of chymostatin, 11) it seems unlikely that such a chymostatin-sensitive protease is essential for sperm penetration through the vitelline envelope in sea urchins under physiological conditions.…”

“…11) This protease is currently proposed to be a vitelline-coat lysin, since the purified enzyme has a vitellineenvelope lytic activity. However, since the vitelline-coat-lytic activity and the acetyl-tyrosine ethyl ester-hydrolyzing activity of the purified enzyme is completely inhibited by low concentrations (100-200 mM) of chymostatin, 11) it seems unlikely that such a chymostatin-sensitive protease is essential for sperm penetration through the vitelline envelope in sea urchins under physiological conditions. Further studies on the effects on fertilization of specific inhibitors and antibodies against the 19-kDa sperm protease and the sperm proteasome, as well as on the localization of these proteases are necessary to convincingly demonstrate the roles of sperm proteases in fertilization.…”

“…10) A 19-kDa chymotrypsin-like protease, which was purified from sea urchin sperm, is thought to be a lysin, since the purified preparation has a vitelline-coat lytic activity. 11) On the other hand, the sperm proteasome has been proposed to participate in the acrosome reaction, since chymostatin and Suc-Leu-Leu-ValTyr-MCA, a preferred substrate for the proteasome, are capable of inhibiting the acrosome reaction in sea urchins. 12,13) However, the precise roles of sperm proteasomes in fertilization, by using several proteasome-specific inhibitors, have not been tested.…”

IV-3 Fertilization

“…Using several protease inhibitors, Hoshi et al (1979) showed that a chymotrypsinlike protease is presumed to be a lysin of the sea urchin, Hemicentrotus pulcherrimus. From spermatozoa of sea urchins, Yamada and Aketa (1981) extracted a factor which dissolves the vitelline layer of the egg with chymotrypsin-like activity, and they purified it (Yamada, Matsui and Aketa, 1982). The enzyme digested the jelly coat and vitelline layer and was released from the acrosome-reacted spermatozoa.…”