Studies on the Egg-Membrane Lysin of Tegula Pfeifferi: Quantitative Assay of the Lytic Activity

Haino, K.; Kigawa, M.

doi:10.1111/j.1440-169x.1969.00203.x

Cited by 12 publications

(4 citation statements)

References 7 publications

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“…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].…”

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confidence: 99%

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

Yamada

Matsui

Aketa

1982

European Journal of Biochemistry

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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...

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

Yamada

Matsui

Aketa

1982

European Journal of Biochemistry

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“…discus . The lytic activities of these purified VC lysins on the homologous VCs have been assayed mainly by 1) elevation and/or disappearance of the VCs from the oocyte surfaces, 2 ) a change in turbidity of VC suspensions as detected by the method developed by Haino and Kigawa (1969), or 3) the change in volume of isolated VCs as detected by the method of Ogawa and Haino-Fukushima (1984) following VC-lysin treatment. Only in Turbo have the effects of the isolated VC lysin on VC morphology been examined directly in thin sections, revealing that "the fibrous structure constituting" the Turbo VC became "coarser than that of the control" after incubation with the :ysin.…”

Section: Discussionmentioning

confidence: 99%

Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone, Haliotis discus

Usui

Haino-Fukushima

1991

Molecular Reproduction Devel

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Two proteins of molecular weights 20,000 (20K) and 15,500 (15.5K) are the major soluble substances released from the acrosomal vesicle of the abalone, Haliotis discus, spermatozoon. A crude preparation of them has been shown to possess lytic activity on the oocyte vitelline coat (VC). To elucidate the role(s) of each acrosomal protein (AP) in VC lysis, oocytes were examined after treatment with various AP preparations. The VC, which is about 1 micron thick, is composed of thin outer and inner electron-dense layers and a thick main layer of a fine filamentous feltwork. When oocytes were treated with a crude preparation containing both APs, the outer layer disappeared and the feltwork of the main layer loosened extensively. A preparation containing predominantly the 20K AP dissolved the outer layer completely and the main layer to some extent, whereas another preparation containing predominantly the 15.5K AP caused loosening of the main layer without alteration of the outer layer, suggesting that the 20K AP acts on the outer layer, whereas the 15.5K AP acts on the main layer. However, when purified, each AP by itself failed to dissolve the VC, although lysis occurred in a 1:1 mixture of these preparations. Moreover, when the oocytes were pretreated with the 20K AP and thoroughly washed, the 15.5K AP alone could induce lysis. These results suggest that the lysis of the outer layer requires both APs but not simultaneously. The 15.5K AP, which is located posteriorly in the acrosomal vesicle, must be released to act on the VC following the action of the 20K AP.

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“…We examined whether the present factor has the vitelline layer lytic activity. The activity can be estimated by the turbidimetric method [Haino and Kigawa, 1969;Heller and Raftery, 1973;Yamada and Aketa, 19811 or by the observation of ultrastructural changes of the egg surface (eg, elongation of microvilli) [Yamada and Aketa, 1981;Yamada et al, 19821. Our factor did not decrease the turbidity of the vitelline layer suspension: The treatment of jellyless eggs with our factor caused no drastic changes such as elongation of microvilli.…”

Section: Discussionmentioning

confidence: 99%

A species‐specific sperm factor dispersing the jelly coat of the egg of the sea urchin, Anthocidaris crassispina

Yamada

Aketa

1983

Gamete Research

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A species-specific factor capable of dispersing the jelly coat surrounding eggs has been purified from sperm of the sea urchin, Anthocidaris crassispina. It does not exert its effect on the vitelline layer. The purification has been accomplished by a four-step procedure involving ammonium sulfate fractionation, gel filtration on Sepharose CL-4B, ion-exchange column chromatography on DEAE-cellulose, and affinity column chromatography on heparin-Sepharose CL-6B. The isolated factor is homogenous in sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 0-rnercaptoethanol, estimated molecular weight being about 140,000.The jelly dispersion by the present factor is activated by CaCI2, and inhibited by KCI, MnCI2, EDTA, and EGTA, and by sulfated saccharides such as chondroitin sulfate A and C, heparin, and glucose-6-sulfate. Inorganic sulfates such as (NH&S04 and Na2S04 have no effect on jelly dispersion. This factor is heat-labile, losing its activity in 30 min at 50°C.The present factor is found also in the seminal plasma, and released from sperm themselves by treatment with Triton X-100. These results suggest that this factor is loosely bound to the sperm surface. Although glycosidase and arylsulfatase activities are detectable in the seminal plasma, these enzyme activities are not detectable in the purified jelly dispersing factor.Only trypsin and or-chymotrypsin among commercial enzymes tested disperse the jelly coat, but neither activity is detectable in our preparation. The jelly-dispersing activity is inhibited neither by trypsin inhibitors such as N-a-p-tosyl-L-lysine-chloromethyl ketone, soybean trypsin inhibitor, ovomucoid trypsin inhibitor, nor by chymotrypsin inhibitors such as L-I-tosylamide-2-phenyl-ethylchloromethyl ketone and chymostatin. Participation of trypsin-like and chymotrypsin-like enzymes in jelly dispersion seems unlikely.

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Studies on the Egg-Membrane Lysin of Tegula Pfeifferi: Quantitative Assay of the Lytic Activity

Cited by 12 publications

References 7 publications

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

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

Two major acrosomal proteins act on different parts of the oocyte vitelline coat in the abalone, Haliotis discus

A species‐specific sperm factor dispersing the jelly coat of the egg of the sea urchin, Anthocidaris crassispina

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