J. Biochem.116,464-470 (1994)

Purification and Characterization of a Novel -Like from Boar Cauda Epididymal Sperm

Kuniko Akama, Kimio Terao,1Yukiharu Tanaka, Akihiko Noguchi,Naoto Yonezawa, Minoru Nakano, and Tohru Tobita Department of Chemistry, Faculty of Science, Chiba University, Inage-ku, Chiba, Chiba 263

Received for publication, April 18, 1994

A -like protease was extracted with 1% cetyltrimethylammonium bromide (CTAB) at pH 7.0 from boar cauda epididymal sperm nuclei whose acrosin had previously been removed by acid extraction. The CTAB-extracted sperm protease (CSP) was purified by ion-exchange chromatography on CM-23, gel filtration on Sephadex G-100, affinity chro matography on benzamidine-CH-Sepharose 4B, and HPLC on CM-5PW. CSP is a two chain protein composed of Mr 2.6K and Mr 37K chains, which are covalently cross-linked by disulfide bonds. CSP exhibited a pH optimum between pH 8.0 and 9.0, and was inhibited by diisopropyl phosphorofluoridate, antipain, leupeptin, and 1-chloro-3-tosylamide-7-amino - L-2-heptanone. The activity of CSP was enhanced about 1.2-fold with 50mM CaCl2, with which acrosin is enhanced 2.0-fold. The catalytic efficiency (kat/KR,) of CSP toward Bz L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe in the presence of 50mM CaCl2 differed from that of acrosin by factors of 0.53, 1.2, and 0.80, respectively. Amino acid sequencing of V8-digested peptides of CSP, and its L and H-chains showed that the amino acid sequence of CSP was closely related to, but different from, that of acrosin. These results suggest that CSP is a novel acrosin-like enzyme that differs from acrosin in its location in the sperm head, the effect of calcium ions on its activity, and its substrate specificity.

Key words: acrosin-like, protease, sperm.

The acrosome, a modified lysosome surrounding the ante

rior part of the sperm head, contains various hydrolytic EXPERIMENTAL PROCEDURES . Acrosin [EC 3.4.21.10] is one of the released as a consequence of the acrosomal reaction, and is Materials•\Bz-L-Arg-OEt, Tos-L-Arg-OMe, Tos-L-Lys classified as a member of the superfamily - OMe, Bz-L-Ala-OMe, and [L-3-trans-carboxyoxiran-2-car (1, 2). Acrosin has been believed to be responsible for the bonyl]-1-Leu-agmatin (E-64) were purchased from Pep limited hydrolysis of the glycoprotein matrix of the egg tide Laboratory (Osaka). Bz-L-Leu-OEt was synthesized by zona pellucida, by which sperm penetration is facilitated the method of Ottesen and Spector (11). Diisopropyl (3-5). However, it has been suggested that mammalian phosphorofluoridate (DFP),1-chloro-3-tosylamide-7-amino acrosins do not or only slightly digest the zona pellucida of L-2-heptanone (TLCK), and p-amino benzamidine dihy the ovum from the same species of mammal (6-10). Recent drochloride were from Sigma Chemical. CM-23 was from findings suggest that (pro) acrosin plays an important role in Whatman. Sephadex G-100 and CH-Sepharose 4B were sperm-egg recognition and binding during the initial stages products from Pharmacia Fine Chemicals. CM-5PW and of fertilization (6-9). In this paper, we describe the Nucleosil 300 7C18 were from Toso and Chemco, respec purification and characterization of another acrosin-like tively. 4-Methylumbelliferyl p-guanidinobenzoate and enzyme from boar cauda epididymal spermatozoa, which 4-methylumbelliferone were from Sigma. Staphylococcus differs from acrosin in acid solubility, kinetic properties, aureus V8 protease [EC 3.4.21.19] (V8) was from Wako and chromatographic behavior on CM-5PW and Nucleosil Pure Chemical Industry. The reagents and solvents for gas 300 7C18. phase sequence analysis were purchased from Applied Biosystems Japan. 1 Present address: Central Research Laboratories, Chugai Pharma Benzamidine-CH-Sepharose 4B was prepared as follows. ceutical Co., Ltd., Toshima-ku, Tokyo 171. CH-Sepharose 4B (5g) was converted to the N-hydroxy Abbreviations: Buffer A, 0.1M NaCl/0.005% Brij 35/50mM acetate succinimide ester, which was then coupled with p-amino buffer, pH 5.5; CSP, CTAB-extracted sperm protease from boar benzamidine-HCl (1g) by the method of Cuatrecasas and cauda epididymis; CTAB, cetyltrimethylammonium bromide; DFP, Parikh (12). diisopropyl phosphorofluoridate; DIP, diisopropylphosphoryl; E-64, Enzymatic Assays•\Esterase activity toward Bz-L-Arg [L-3-traps-carboxyoxiran-2-carbonyl]-1-Leu-agmatin; L-chain, - OEt was measured by the method of Schwert and Takenaka light chain; H-chain, heavy chain; PCMPS, p-chloromercuriphenyl sulfonic acid; SBTI, soybean trypsin inhibitor; TLCK, 1-chloro-3 (13) at 25•Ž. Unless otherwise stated, the assay was tosylamide-7-amino-L-2-heptanone; V8, Staphylococcus aureus V8 performed in 0.1M Tris-HCl buffer, pH 8.0. One enzyme protease. unit was expressed as the transformation of 1ƒÊmol of

464 J. Biochem. A Novel Acrosin-Like Enzyme from Sperm 465

substrate per min under the conditions described. The (2.6•~95 cm) equilibrated with buffer A. The active frac hydrolysis of Tos-L-Arg-OMe and Tos-L-Lys-OMe was tions were subjected to affinity chromatography on benzam followed at 247 nm with the use of a molar absorption idine-CH-Sepharose 4B (Fig. 2). The active fractions difference of 533M-1•Ecm-1, and that of Bz-L-Arg-OEt at eluted from benzamidine-CH-Sepharose 4B were adjusted 253nm with 1,150M-1•Ecm-1, Bz-L-Leu-OEt at 256nm to pH 5.5, and then chromatographed on CM-5PW (Fig. 3). with 222M-1•Ecm-1, and Bz-L-Ala-OMe at 256nm with 964 Isolation of Acrosin from Boar Cauda Epididymal M-1•Ecm-1 (14-16). Acrosin activity toward Bz-L-Arg-OEt Sperm-The acid-extracted acrosin from cauda epididymal was assayed in 50mM CaCl2/0.1M Tris-HCl, pH 8.0, by a sperm was precipitated with ethanol, extracted with buffer modification of the method of Schleuning and Fritz (17) . A, and then chromatographed by a modification of the Specific activity was expressed in units per mg of protein. method of M.-Esterl et al. (18). After chromatography on Determination of Protein-The protein concentrations in benzamidine-CH-Sepharose 4B, the acrosin was further crude materials were determined by measurement of the purified by chromatography on CM-5PW (Fig. 3). absorbance at 280 nm. Protein was estimated by assuming Determination of the Operational Molarities of CTAB that 1 mg of protein per ml exhibits an absorbance value of Extracted Protease and Acrosin•\The titration of CTAB 1.0 at 280 nm with a 1 cm light path. The amounts of the extracted protease and acrosin with 4-methylumbelliferyl

purified protein and component peptide were determined p-guanidinobenzoate was performed by a modification of by amino acid analysis. the method of Brown et al. (19) in 0.1 M sodium barbi Collection of Cauda Epididymal Sperm•\Unless other turate-HCl buffers, pH 8.2, containing 0-1.0M CaCl2, wise specified, all operations in the following experiments diluted with 0.2 volumes of dimethyl sulphoxide at 20•Ž. were performed at 4•Ž. Boar cauda epididymides (400 g) One hundred sixty to 1,280mU (substrate, Bz-L-Arg-OEt) were cut into slices with a knife and then suspended in 0.15 of CTAB-extracted protease or 290-2,320mU (substrate, M NaCl (800 ml). The suspension was stirred for 30 min, Bz-L-Arg-OEt) of acrosin was put into a 3-ml cuvette and then filtered through two layers of gauze and a stainless containing 500-1,000 pmol/ml of 4-methylumbelliferyl steel mesh (63 km). The filtrate was centrifuged at 1,500•~ p-guanidinobenzoate. The fluorescence of 4-methylumbel g for 15 min. The sperm pellet was resuspended in 0.25 M liferone was monitored for 8 min with a Hitachi 650-50 sucrose/0.15 M NaCl/10mM Tris-HCl, pH 8.0 (400 ml), spectrophotofluorimeter with wavelength settings of 363 and then sedimented againainnnnnnnnnnnby centrifugation. nm (excitation) and 457 nm (emission). Ac Ac Ac Acidand Cetyltrimethylammonium Bromide-Extraction Inactivation and S Carboxymethylation of CTAB-Ex of Proteases from Cauda Epididymal Sperm-Acrosin was tracted Protease and Acrosin•\The active enzyme frac extracted with acid by a modification of the method of tions eluted from the benzamidine-CH-Sepharose 4B M.-Esterl et al. (18) from boar cauda epididymal sperm. column were adjusted to pH 8.5 and then treated with 3.3 This extraction was repeated five times, acrosin being mM DFP at room temperature overnight. The diisopropyl thereby removed thoroughly (Fig. 1). The pellet of acrosin phosphoryl (DIP) enzyme was chromatographed on CM depleted sperm was suspended in 1.62 M sucrose/50mM 5PW by the same procedure as for the active enzyme (Fig. acetate buffer, pH 6.0 (200 ml), with a teflon homogenizer, 3) to remove excess DFP and autolytic peptides of the and then centrifuged at 10,000•~g for 10 min for removal enzyme, if any. The DIP-enzyme fraction from CM-5PW of mitochondria and other intracellular organelles. This was subjected to HPLC on Nucleosil 300 7C18 (Fig. 5, A treatment was repeated twice. The pellet was washed with and B) to remove salts, and then lyophilized. 0.31 M sucrose/50mM acetate buffer, pH 6.0 (100 ml). Isolation of Light and Heavy Chains of CTAB-Extracted The washed pellet was suspended gently in 1% cetyltri Protease and Acrosin•\The DIP-enzyme was reduced and methylammonium bromide (CTAB)/50mM triethanol then carboxymethylated by a modification of the method of amine-NaOH buffer, pH 7.0, with a teflon homogenizer, Crestfield et al. (20). The reduced and carboxymethylated and then the suspension was stirred for 10 min at 25•Ž, and protein was subjected to HPLC on Nucleosil 3007C18 (Fig. centrifuged at 12,000•~g for 10 min. This treatment was 5, C and D). The peptide in each peak was recovered by repeated four times (Fig. 1). lyophilization and then redissolved in 0.1% trifluoroacetic Isolation of CTAB-Extracted Protease•\The combined acid. Aliquots were used for amino acid and N-terminal CTAB-extract was precipitated with 6 volumes of ethanol amino acid sequence analyses. at -20•Ž for 1 h. The precipitate was recovered by centri Amino Acid Analysis-Samples were hydrolyzed in fugation at 12,000•~g for 20 min. The pellet was extracted vacuo with 6M HCl at 110•Ž for 24 h. Amino acids were three times with 0.1M NaCl/0.005% Brij 35/50mM determined with a Hitachi 655 amino acid analyzer. acetate buffer, pH 5.5 (buffer A). The insoluble material Electrophoresis-SDS-PAGE was performed at pH 8.8 was removed by centrifugation at 12,000•~g for 20 min. on a 15% gel by the method of O'Farrell (21). The gel was For the following procedures, the glass vessels were silicon stained with a silver-based stain (22). ized before use, and plastic tubes and beakers were used to Amino Acid Sequencing of Peptides•\The light (L-) and avoid protein adsorption to the surface of glass. heavy (H-) chains of CTAB-extracted protease (300 kg) The extract with buffer A was subjected to ion-exchange were sequenced with a sequencer (470A protein sequencer; chromatography on CM-23 (1.9•~30 cm) equilibrated with Applied Biosystems). The DIP-CTAB-extracted protease buffer A. The CTAB-extracted protease was eluted with a or DIP-acrosin (220ƒÊg) was boiled in 2.3% SDS/3.8mM 0.1-0.6 M NaCl linear gradient (total 240 ml). The active EDTA/75mM ammonium bicarbonate buffer, pH 7.8, for fractions were dialyzed against 0.005% Brij 35/10mM 2 min, and then cooled to room temperature. The enzyme acetate buffer, pH 5.5, for 6 h, and then lyophilized. The treated with SDS was digested with V8 (4 jig) for 6 h at lyophilized materials were dissolved in buffer A, and then 37•Ž. The digest (20 jig for the peptide map and 200 jig for the solution was applied to a column of Sephadex G-100 amino acid sequencing of peptides) was subjected to two

Vol. 116, No. 2, 1994 466 K. Akama et al.

dimensional SDS-PAGE (2D SDS-PAGE). SDS-PAGE in found to have decreased by 2.0-fold. In contrast, the the first dimension was performed under the standard CTAB-extracted activity increased by 1.2-fold in the conditions described above. The gel was soaked in the presence of 50mM CaCl2. When the acid and CTAB reducing sample buffer for 30 min. The gel was then placed - extracts were incubated at pH 5.3 by the method of on the second 15% gel and fixed with 1% agarose. The Polakosky and Zaneveld (24) to activate proacrosin to second electrophoresis was performed in the orthogonal acrosin, the activity of the acid extract increased about direction with the same electrophoresis buffer as used for 1.7-fold upon the incubation, but that of the CTAB-extract the first dimension. After 2D SDS-PAGE, the peptides remained unchanged, indicating that proacrosin could be were stained with a silver-based stain (Fig. 6) or electrob extracted with acid. These results showed that boar cauda lotted onto polyvinylidene difluoride (PVDF) membranes epididymal sperm had another protease, that differed from by a slight modification of the method of Hirano (23) using acrosin in acid-solubility and the effect of calcium ions on its transfer buffer containing 0.05% SDS, stained with Co activity. Thus, we named this protease, CTAB-extracted omassie Brilliant Blue, and then sequenced directly with a sperm protease (CSP), and purified and characterized CSP sequencer (Fig. 7). in comparison with acrosin. Purification of CSP and Acrosin•\CSP and acrosin were

RESULTS purified through five steps from CTAB and acid-extracts, respectively. The results of their purification are summa Extraction of a Protease with CTAB from Acrosin-Free rized in Tables I and II. CSP and acrosin showed similar Sperm Nuclei•\Several conditions were examined for the chromatographic behavior on CM-23, Sephadex G-100, and extraction of an enzyme hydrolyzing Bz-L-Arg-OEt from benzamidine-CH-Sepharose 4B (Fig. 2). However, CSP boar cauda epididymal sperm nuclei, whose acrosin had could be separated from acrosin by HPLC on CM-5PW been removed by acid-extraction. The most suitable agent (Fig. 3). CSP and acrosin emerged at 0.88 and 0.96M NaCl for extraction of the enzyme from the nuclei was 1% CTAB/ from the CM-5PW column, respectively. CSP and acrosin 50mM triethanolamine-NaOH buffer (pH 7.0). However, obtained on HPLC on CM-5PW were too labile for their 10mM 3-[(3-cholamidopropyl)dimethylammonio]-1-pro properties to be examined. Both enzymes were stabilized in panesulfonate/20% glycerol/100mM triethanolamine the presence of Brij 35 at a concentration of 0.005%. The NaOH, pH 7.0, was less effective, and 2.5M NaCl/50mM highest specific activity towards Bz-L-Arg-OEt was 240 for acetate buffer, pH 4.0, and 2.5M NaCl/50mM sodium CSP and 250 for acrosin. The yields of CSP and acrosin phosphate buffer, pH 7.4, had no effect at all. were 1.2 and 2.0 mg, respectively, per 400 g of cauda Figure 1 shows the results of the seven-times acid epididymides. DIP-CSP and DIP-acrosin each migrated as extraction and the four-times CTAB extraction of the a somewhat broad band of 39kDa under nonreduced enzymes hydrolyzing Bz-L-Arg-OEt from boar cauda epidi conditions, and as a single band of 37kDa under reduced dymal sperm. The results of the acid extraction indicate conditions on SDS-PAGE (Fig. 4). These values were well that the activity and specific activity of each extract consistent with those of boar ƒÀ-acrosin from ejaculated decreased with increasing number of extraction times. The sperm (25-30). enzymatic activity after the first CTAB extraction was Enzymatic Properties of CSP-CSP and acrosin showed higher than that after the last acid extraction. The ratios of the highest activity around pH 8.0-9.0. Table III shows the the acid and CTAB-extracted activities to the total activity were 0.60 and 0.40, respectively. When assayed in the absence of 50mM CaCl2, the acid-extracted activity was TABLE I. Summary of purification of CSP. CTAB-extract, extract with 1% cetyltrimethylammonium bromide/50mM trieth anolamine-NaOH buffer, pH 7.0; Benzad-CH-Seph 4B, benzamidine CH-Sepharose 4B.

TABLE II. Summary of purification of boar sperm acrosin from cauda epididymis. Phenyl-Seph 4B, Phenyl-Sepharose 4B.

Fig. 1. Sequential extraction of hydrolytic activity towards Bz-L-Arg-OEt with acid and CTAB from boar cauda epididymal sperm. _??_, hydrolytic activity of the acid-extract in the presence of 50mM CaCl2; _??_, hydrolytic activity of the acid-extract assayed in the presence of 50mM CaCl2 after incubation at pH 5.3 by the method of Polakosky and Zaneveld (24); M, hydrolytic activity of the CTAB-extract in the absence of 50mM CaCl2.

J. Biochem . A Novel Acrosin-Like Enzyme from Sperm 467

effects of various protease inhibitors on the activities of CSP and acrosin. CSP as well as acrosin was strongly inhibited by DFP, antipain, leupeptin, TLCK, soybean trypsin inhibitor, and benzamidine, but not or only partially by p-chloromercuriphenyl sulfonic acid (PCMPS), EDTA, E-64, chymostatin, and elastatinal. However, CSP was partially inhibited by pepstatin and bestatin, by which acrosin was not. These results suggest that CSP as well as acrosin may be characterized as a serine protease with serine and histidine residues in its active center (31, 32). The operational molarities of CSP and acrosin were determined by the use of 4-methylumbelliferyl p-guani dinobenzoate. The titration of CSP and acrosin in buffers

Fig. 4. SDS-PAGE of CSP and acrosin. Lanes 1 and 2, DIP-CSP and acrosin in the presence of 2-mercaptoethanol, respectively. Lanes 3 and 4, DIP-CSP and acrosin in the absence of 2-mercaptoethanol, respectively. The purified protein (4ƒÊg) was electrophoresed in each lane. The mobilities of molecular-mass markers (rabbit muscle

phosphorylase b, bovine serum albumin, hen egg white ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor, and hen egg white lysozyme), in kDa, are indicated by arrowheads.

TABLE III. Effects of protease inhibitors on CSP and acrosin. CSP (0.42ƒÊg) or acrosin (0.42ƒÊg) in 100 pl was incubated with each protease inhibitor in 0.005% Brij 35/0.1M Tris-HCl, pH 8.0, with (for acrosin) or without (for CSP) 50mM CaCl2 for 60 or 10 min at 25•Ž, and then the residual enzyme activity was assayed using Bz L-Arg-OEt as the substrate in the same buffer as described above. PCMPS, p-chloromercuriphenyl sulfonic acid; SBTI, soybean trypsin inhibitor.

Fig. 2. Affinity chromatography of CSP on benzamidine-CH Sepharose 4B. The CSP fractions (1 mg) eluted from Sephadex G-100 were chromatographed on a column (0.8•~3.5cm) of benzam idine-CH-Sepharose 4B. After the column had been washed with 1M NaCl/0.1% Brij 35/0.2M triethanolamine, pH 7.8, and successively washed with 0.1M NaCl/0.005% Brij 35/50mM acetate buffer, pH 5.5, the adsorbed material was eluted with 0.1M NaCl/0.1% Brij 35/ 0.1M glycine-HCl, pH 3.0. Fractions (3.5 ml) were collected at the flow rate of 15 ml/h.

TABLE IV. Kinetic parameters as to three ester substrates of CSP and acrosin. A mixture, in a volume of 3.0 ml, containing 30 350ƒÊM substrate and 0.005% Brij 35/0.1M Tris-HCl, pH 8.0, with or without 50mM CaCl2 was incubated at 25•Ž, and then the reaction was started by the addition of 50,u l of enzyme solution. Km and kcat, were estimated with the equation: s/v0=1/kcat,[E](Km+[S]0) (42). +Ca2+ or -Ca2+: with or without 50mM CaCl2; MS-1, (10-3 •~ s)-1.

Fig. 3. HPLC of CSP and acrosin on CM-5PW. The active fractions [(A) 0.48 mg of CSP, (B) 0.7 mg of acrosin] eluted from benzamidine-CH-Sepharose 4B were chromatographed on a column (0.75•~7.5cm) of CM-5PW with a 0-1.2M NaCl linear gradient in 0.005% Brij 35/20mM acetate buffer, pH 5.5, at 21•Ž. Fractions (1.0 ml) were collected at the flow rate of 1.0 ml/min.

Vol. 116, No. 2, 1994 468 K. Akama et al.

containing 0, 0.05, and 1.0M CaCl2 showed that the burst marized in Table IV. CSP and acrosin exhibited the highest rate was essentially the same in buffers with various CaCl2 catalytic efficiency with Tos-L-Arg-OMe and Bz-L-Arg concentrations. The molarity of the enzyme solution was - OEt, respectively. The catalytic efficiency (kcal/Km) of CSP calculated to be 2.31nmol/ml for 31.1 units/ml of CSP and as to Bz-L-Arg-OEt, Tos-L-Arg-OMet, and Tos-L-Lys-OMe 3.79nmol/ml for 59.4 units/ml of acrosin from the initial in the presence of 50mM CaCl2 differed from that of burst in the buffer containing 50mM CaCl2, which means acrosin by factors of 0.53, 1.2, and 0.80, respectively. The that 1 mol of CSP and acrosin hydrolyse 13,500 and 15,700 kcat values of CSP and acrosin for Bz-L-Arg-OEt increased mol Bz-L-Arg-OEt per min, respectively, under the condi by 1.2 and 2.0-fold, respectively, with 50mM CaCl2. tions employed. EDTA (2mM) did not affect the activity of either CSP or The kinetic properties of CSP and acrosin as to Bz acrosin (Table III), indicating that the presence of Ca" was L-Arg-OEt, Tos-L-Arg-OMe, and Tos-L-Lys-OMe are sum- important but not indispensable for the activities of CSP and acrosin. Bz-L-Ala-OMe and Bz-L-Leu-OEt were not hydrolyzed by CSP and acrosin at all. Isolation of the Peptide Chain Components of CSP •\ CSP as well as acrosin was a two-chain protein whose L and H-chains were separated by HPLC on Nucleosil 300 7C18 after reduction and carboxymethylation of the enzyme (Fig. 5) : The L-chains of CSP and acrosin were eluted from the column with 26 and 32% acetonitrile, respectively, whereas the H-chains of CSP and acrosin were eluted with 48 and 45% acetonitrile, respectively. The whole CSP and acrosin before reduction and carboxymethylation were eluted at 45 and 50% acetonitrile, respectively. The amino acid compositions of CSP and acrosin, and their L and H-chains resembled each other (Table V). The Mr of the L-chain of CSP was estimated to be 2.6K on the basis of the amino acid composition. Digestion of CSP and Acrosin with V8, and Partial Amino Acid Sequence Determination of CSP-DIP-SDS

TABLE V. Amino acid compositions (molar ratio) of CSP and acrosin, and their H and L-chains.

Fig. 5. Reverse-phase HPLC of CSP and acrosin, and their light and heavy chains. The DIP-enzyme (0.49 mg of CSP or 0.56 mg of acrosin) was chromatographed on a column (4•~150mm) of Nucleosil 300 7C18 in 0.1% trifluoroacetic acid with a 0-80% acetonitrile (MeCN) linear gradient at 40•Ž. Fractions (1.0 ml) were collected at the flow rate of 1.0 ml/min. (A) native CSP, (B) native acrosin, (C) reduced and carboxymethylated CSP, (D) reduced and carboxymethylated acrosin.

Fig. 6. Two-dimensional SDS PAGE of V8-digested peptides of CSP and acrosin. For the first and second-dimensions, SDS-PAGE was

performed in 15% gels under non reducing and reducing conditions, respectively. The digest (20ƒÊg) was applied, and the gel stained with a silver-based stain. (A) The digest of CSP, (B) the digest of acrosin.

J. Biochem. A Novel Acrosin-Like Enzyme from Sperm 469

acrosin can be characterized as a trypsin-like serine pro tease exhibiting susceptibility to DFP, antipain, leupeptin, TLCK, soybean trypsin inhibitor, and benzamidine. CSP closely resembles acrosin in optimal pH and sensitivity to some kinds of inhibitors (Table III), and is also a two chain protein composed of L and H-chains, which are covalently cross-linked by disulfide bonds (Fig. 5), the amino acid compositions of the two peptide chains resembling those of acrosin (Table V). However, CSP showed clear differences from acrosin in acid-solubility (Fig. 1), chromatographic behavior on CM-5PW (Fig. 3), and Nucleosil 300 7C18

(Fig. 5), the effect of calcium ions on its activity, and kinetic properties (Table IV). Amino acid sequencing of the L and H-chains of CSP, and peptides 1-3 obtained on V8-diges tion of DIP-SDS-CSP (Fig. 7) showed that the amino acid sequence of CSP was closely related to, but different from, that of acrosin. The presence of enzymes like CSP has never previously been reported. The Km values of boar acrosin for Bz-L-Arg-OEt (90ƒÊM in 0.005% Brij 35/0.1M Tris-HCl, pH 8.0, and 101ƒÊM in 0.005% Brij35/50mM CaCl2/0.1M Tris-HCl, pH 8.0) (Table IV) were intermediate between the previously reported Km value of 270ƒÊM in 45mM triethanolamine - HCl, pH 8.0 (26), and that of 50ƒÊM in 50mM CaCl2/50 mM Tris-HCl, pH 8.0 (14). The discrepancies between our results and the published Km values may be due to the differences in the acrosin preparation and assay conditions. The purified CSP as well as acrosin was stabilized by the addition of the non-ionic detergent, Brij 35, at a concentra Fig. 7. N-terminal amino acid sequences of V8-digested pep tion lower than the critical micelle concentration. Similar tides of CSP. Peptide 1 means the peptide in spot 1 in Fig. 6A, etc. stabilizing effects of non-ionic detergents and phospholipids Dashes represent residues not identified. The amino acids identical on the activity of ejaculated boar acrosin have been previ with those of boar acrosin are shown by * between the corresponding ously reported, and acrosin has been suggested to be an letters. The numbering is that for boar proacrosin (40, 41). These extrinsic membrane protein containing the hydrophobic sequences were compared using the GENETYX program. The consen sus sequence of acrosins (inv) is derived from Klemn et al. (39). area responsible for its interaction with membranes (35). The affinity of acrosin to artificial and natural membranes indicates the association of the protease with the acrosomal CSP and DIP-SDS-acrosin were digested with V8, which is membrane (36-38). However, Klemn et cal. (39) have known to hydrolyze the carboxyl side of glutamic acid pointed out the following on the basis of the available residues in proteins in a specific manner in ammonium acrosin sequences (39, 40): Of two notable hydrophobic bicarbonate buffer (33). The digest was analyzed by 2D segments in mammalian acrosin sequences, the one at the SDS-PAGE (Fig. 6). The mobilities of spots 1-4 for CSP amino-terminus representing the putative signal peptide is (Fig. 6A) were slightly faster than those for acrosin (Fig. cleaved off before acrosin enters the acrosome, and the 6B) on SDS-PAGE under reducing conditions (second other in the vicinity of the (serine) is also present dimension). Spots 1 and 2 for CSP disappeared more slowly in other serine proteases not associated with biological on V8-digestion than those for acrosin (data not shown). membranes. That is, the structural basis of the interaction The N-terminal amino acid sequences of V8-digested of acrosin with biological membranes, a prerequisite for peptides of CSP, and its L and H-chains are shown in Fig. zona binding and penetration (41), remains to be explained. 7. The amino acid sequence of the L-chain of CSP and the CSP was only efficiently extracted with the ionic detergent, N-terminal sequence of the H-chain of CSP were the same CTAB, at pH 7.0, i, e., it was not extracted with acid (Fig. as those of acrosin. The N-terminal sequences of peptides 1 1). These findings suggest that CSP is different from and 2 exhibited 62 and 71% homology with residue Nos. acrosin in its location in the sperm head: There is a 116-128 and 184-200 of acrosin, respectively. The N possibility that CSP is integrated into the membrane or is terminal sequence of peptide 3 was the same as that of the located inside the membrane of the nucleus. Further H-chain of CSP. The sequence of peptide 4 was not studies will reveal the possible interactions of CSP and analyzed, since it was only slightly electroblotted onto a acrosin with the membrane in the spermatozoon, and the PVDF membrane under the conditions used. relationship between their structures and functions.

DISCUSSION The authors are indebted to Professor Koscaku Maruyama and Dr. Sumiko Kimura of Chiba University for the fluorescence spectro The properties of acrosin from boar cauda epididymal photometric analysis. sperm were almost the same as those of boar f3-acrosin from ejaculated sperm (14, 18, 25-30, 34). CSP as well as

Vol. 116, No. 2, 1994 470 K. Akama et al.

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