HUMAN ACROSIN: PURIFICATION AND SOME PROPERTIES
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T. KOBAYASHI, Y. MATSUDA, S. OSHIO, S. KANEKO, S . NOZAWA, H. MHORI, S . AKIHAMA, and Y. FUJIMOTO
Human sperm with normal morphology and good viability were obtained by centrifugation using a discontinuous Percoll density gradient with an inner column. Acrosin (E.C.3.4.21.10) was rapidly purified from sperm by ion exchange adsorption and elution and was purified by affinity adsorption on a lima bean trypsin inhibitor (LBTI) Cellulofine column. The final preparation was found to be homogeneous on polyacrylamide gel electrophoresis and to have a molecular weight of about 4 x lo4 daltons. The enzyme had an esterolytic activity of 3.5 pnol/min/A280 with N-a-tosyl-L-arginine methyl ester as the substrate. Human acrosin showed a broad substrate specificity for arginine and lysine derivatives and it seemed to have a somewhat different specificity from trypsin. The optimal pH of this enzyme with amidolytic activity was 9.0. Enzyme activity was stimulated by a high concentration of calcium chloride. LBTI and aprotinin strongly suppressed the amidolytic activity with the o-valyl-Lleucyl-L-arginine-p-nitroanilide(Val-Leu-Arg-pNA) as the substrate, but cr,-antitrypsin and soybean trypsin inhibitor were less effective. Key Words: Acrosin; Human seminal plasma; Purification; Affinity chromatography; Substrate specificity; Arginine esterase.
INTRODUCTION Acrosin (E.C. 3.4.21.10) has been reported to be one of the trypsin-like serine proteinases associated with the acrosomal membrane of sperm. The functional role of acrosin in fertilization may be dissolution of the zona pellucida (1 1). Thus, the study of acrosin in human sperm is likely to yield important information on the mechanism of fertilization. Acrosin was first described in rabbit epididymal spermatozoa and mammalian acrosins were later purified by many investigators [4, 7, 12, 131. Human acrosins have been purified using washed sperm prepared by centrifugation of diluted semen. However, the washed sperm could not be freed from contamination with immature and abnormal sperm, other cellular
Received December 11, 1990. Accepted January 16, 1991. Department of Obstetrics and Gynecology, School of Medicine, Keio University (T.K., S.K., S.N.), Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. First Department of Biochemistry, Meiji College of Pharmacy (Y.M., S.A.), Nozawa, Setagaya-ku, Tokyo 154, Japan. Department of Biology, College of Arts and Sciences, University of Tokyo ( S . O . , H.M.), Komaba, Meguro-ku, Tokyo 153, Japan. Department of Clinical Biochemistry, Hokkaido Institute of Pharmaceutical Sciences (Y.F.), Otaru-Shi, Hokkaido 047-02, Japan. Address correspondence to: Dr. Y. Matsuda, First Department of Biochemistry, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo, Japan. ARCHIVES OF ANDROLOGY 21:9-16 (1991) Copyright 0 1991 by Hemisphere Publishing Corporation
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components, and seminal plasma, so morphologically purified sperm preparations are required for accurate biochemical analysis and enzyme purification. Recently, we developed a Percoll density gradient centrifugation method using an inner column for the preparation of human sperm [7] that is capable of isolating motile sperm of normal morphology and decreasing seminal plasma to less than of that in original semen. In the present study we report the rapid purification of human acrosin and some of its properties using morphologically purified human sperm. Syst Biol Reprod Med Downloaded from informahealthcare.com by McMaster University on 11/14/14 For personal use only.
MATERIALSIMETHODS Purified Human Sperm Morphologically purificd human sperm were obtained using a discontinuous Percoll density gradient and inner-column centrifugation as described previously by Kaneko ct al. [7] Viable motile sperm in the sediment were resuspended in saline and werc frozen.
Enzyme Assay Esterolytic activity was colorirnetrically assayed using methyl ester derivatives as substrates and chromotropic acid at pH 8.0 and 30°C, as described by Moriwaki et al. [lo]. We used N-a-tosyl-Larginine methyl ester (Tos-Arg-Me) esterolytic activity to monitor the purification procedure of acrosin. Amidolytic activity was measured using D-valyl-L-leucyl-L-arginine-p-nitroanilide (Val-Leu-Arg-pNA, S-2266) as a substrate [2] at pH 8.0 and 30°C. The concentration of the esterolytic and amidolytic substrates were 10 and 0.5 mmol/L, respectively. All esterolytic and amidolytic activities were expressed in terms of pmol/L substrate hydrolyzed per minute (pmol/min) .
Protein Concentration The protein concentration was estimated spectrophotometrically assuming that the absorbance of 1mg protein in a 1-cm width cuvette at 280 nm is 1.O.
Reagents The following chemicals and proteins were obtained commercially: Aprotinin, CM-Cellulofine, Cellulofine GCL-300, and Formyl Cellulofine (Scikagaku Kogyo Company, Japan). Tos-Arg-Me, LBTI, bovine serum albumin (BSA), acetyl-glycyl-L-lysine methyl ester (Ac-Gly-Lys-Me), N-a-tosyl-L-lysine methyl ester (Tos-Lys-Me), acetyl-L-phenylalanine methyl ester (Ac-Phe-Me) and carbobenzoxy-Lalanyl-L-glycine methyl ester (CBA-Ala-Gly-Me) (Sigma Chemical Company, USA). N-a-Benzoyl-Larginine methyl ester (Bz-Arg-Me), acetyl-N-a-benzoyl-L-citrullinemethyl ester (Bz-Cit-Me) (Serva Chemical Company, Germany). a,-antitrypsin and soybean trypsin inhibitor (SBTI; Boehringer Mannheim, Germany).
RESULTS Samples of 18 ml of the purified sperm that contained lo9 cells/ml with more than 90% motility (normal morphology) were obtained from 100 ml human mixed semen. The purified human sperm were suspended in HCl solution at pH 2.0, containing 10%glycerin, and were crushed using Polytron (type 10/35). An equal volume of the same solution was added to the crushed sperm and shaken vigorously for 2 h. The extracted solution was centrifuged and the supernatant was dialyzed initially against deionized water for 6 h, and then against 0.01 mmol of phosphate buffer per liter at pH 7.0
11
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Human Acrosin
(buffer A) for 4 h. The dialyzed solution was applied to a CM-Cellulofine column preequilibrated with buffer A, and the column was washed with buffer A. The acrosin was eluted with buffer A containing 0.5 M NaCL, and the resulting solution was adjusted to pH 8.0. The active solution from the above step was applied to an LBTI Cellulofine column and the column was washed with 0.05 M Tris-HC1 buffer at pH 8.0 (buffer B). The first elution was made with buffer B containing 0.5 M CaCl and this preparation contained no Tos-Arg-Me esterolytic activity. The acrosin preparation that eluted with HC1 solution at pH 2.0 showed Tos-Arg-Me esterolytic activity (Fig. 1) and was adjusted to pH 8.0. The specific activity of this preparation was 3.50 and 0.161 pmol/min/A,,, for Tos-Arg-Me esterolytic activity and Val-Leu-Arg-pNA amidolytic activity, respectively. The overall results of the purification of human acrosin are summarized in Table 1. The specific activity was 51 times higher than that of the initial extract and the final preparation represented about 6.7 % recovery of Tos-Arg-Me esterolytic activity calculated from the initial extract. The isolated acrosin was homogeneous on polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 4 x lo4daltons by molecular sieve chromatography using a Cellulofine GCL-300 column. The Val-Leu-Arg-pNA amidolytic activity of acrosin, which was dependent upon pH, was measured using modified Britton-Robinson’s widerange buffer between pH 6.0 to 11.O, and the optimal pH was pH 9.0 (Fig. 2). The effect of calcium chloride on Val-Leu-Arg-pNA amidolytic activity of acrosin was tested (Fig. 3). Its effect on the amidolytic activity of basic human seminal plasma arginine esterase (BHSAE) is illustrated for comparison. Following the addition of 30- and 100-mM calcium chloride, the activity was observed to be 3.1 and 3.2 times higher than that of the control (with no addition of calcium chloride), respectively. However, a final concentration of
9
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a w
u z
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0
0
v)
m
CL IW -
4:
i n
w
0
10
FRACTION
20 NUMBER ( 2 , 5
u3
ml/tube
40 )
FIGURE 1 Lima bean trypsin inhibitor Cellulofine affinity adsorption of human acrosin. A, eluted with HCl solution at pH 2.0. 0 , absorbance at 280 nm;0, Tos-Arg-Me esterolytic activity.
T. Kobayashi et al.
12 TABLE 1 Purification of Human Acrosin
Esterolytic Activity
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Procedure Extract CM-cellulose adsorption and elution Lima bean trypsin inhibitor-Cellulofine affinity adsorption
Protein Recovery (%) 100
I .2 0.13
(%)
Specific Activity (pmol/min/A280)
100 13.1
0.068 0.124
Recovery
6.1
3.50
P.F. 1 1.8 51
P.F.. Purification factor.
more than 100 mM calcium chloride decreased the potentiation. Calcium chloride showed no potentiation effect on BHSAE. The esterolytic and amidolytic activities of enzymatically purified human acrosin on synthetic arginine, lysine, and other amino-acid derivatives used as substrates were examined (Table 2). Comparative data of substrate specificity or boar acrosin and BHSAE (Matsuda et al., 1982, 1986) are also shown in Table 2. Tos-Arg-Me and Bz-Arg-Me were easily hydrolyzed by present acrosin and the ratio of the activity of Tos-Arg-Me to Bz-Arg-Me was about 1. Acrosin did not attack Bz-Cit-Me or CBZ-Ala-Gly-Me. A correlation between the ratios of substrate specificity of Tos-Arg-Me to acrosin and BHSAE (Matsuda et al., 1986) was not observed (Fig. 4). Some proteinase inhibitors were tested on Val-Leu-Arg-pNA amidolytic activity of acrosin at pH 8.0 and 30" as shown in Fig. 5. Strong inhibition of acrosin was observed with LBTI and aprotinin whereas a,-antitrypsin and SBTI were less effective. The acrosin isolated in this study was not inhibited by fractions not adsorbed on an LBTI affinity adsorption column (fractions 3 to 10 in Figure 1).
DISCUSSION Human semen contains not only mature motile sperm, but also poor-quality sperm and other cellular components including bacteria. It is important to obtain morphologically normal, highly purified sperm preparations for the purification of an enzyme such as acrosin and for the accurate determination of its functional role in sperm because contaminants like bacteria are rich sources of enzymes and inhibitors. The isolation procedure used here facilitated the collection of normal, viable, and motile sperm without any contamination from seminal plasma or any other source; the purified acrosin was considered to be from mature sperm only. In the present study, human acrosin was purified from a sperm suspension containing lo9 cells/ ml of 90% motile cells. In addition, the present purification procedure is simpler and more rapid than that previously described by others [5, 151. The primary structure of human proacrosin was described by Baba et al. [3] using a deductive method with cDNA. The molecular mass of human proacrosin was predicted to be 43,860 d; the results of our study showed a similar molecular weight although we did not
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Human Acrosin
0’
13
’
6
8
7
9
10
11
PH FIGURE 2 pH dependence of amidolytic activity of human acrosin. The pH dependence of amidolytic activity of human acrosin was measured using Val-Leu-Arg-pNA as the substrate at various pH in 0.08 M modified Britton-Robinson’s wide range buffer. Activity is expressed as a percentage of that at optimal pH.
>-
-2 I-
3,O
I-
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4 U
0
E: + a
2,o
CT
1,o
0
C
1
3
10
CONCENTRATION OF CaC1,
30 (
mM
100
300
1
FIGURE 3 Effects of calcium chloride on the esterolytic activity of human acrosin and basic human seminal plasma arginine esterase (BHSAE). Human acrosin or BHSAE and the indicated amount of calcium chloride were mixed and then Tos-Arg-Me esterolytic activity was measured. Activity is expressed as a ratio to the control (without calcium chloride). 0 , human acrosin; 0, BHSAE.
T. Kobayashi et al.
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TABLE 2 Esterolytic and Amidolytic Activities of Human Acrosin on Synthetic Arginine, Lysine, and Other Amino Acid Derivatives Used as Substrate
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Present Acrosin Substrate
Activity
Ratio
Tos-Arg-Me Bz-Arg-Me Ac-Phe- Arg-Me Ac-Lys-Me Tos-Lys-Me Ac-Gly-Lys-Me Bz-Cit-Me Ac-Phe-Me CBZ-Ala-Gly-Me Val-Leu- Arg-pNA
3.50 3.46 2.31 1.37 1.33 1.89 0 0 0 0.161
1 0.99 0.66 0.39 0.38 0.54 0 0 0 0.046
BHSAE' Ratio 1 0.20 0.73 0.63 1.09 0.94 0 0 0.02
Boar Acrosin' Ratio 1
1.01 3 3
0.43 1.56 3 3
3
3
3
The concentration of esterolytic and amidolytic substrate was 10 and 1 mM, respectively. All activities are expressed in pmol substrate hydrolyzed per min per A,,o at 30°C and pH 8.0. The ratio of activity is given relative to standard Tos-Arg-Me. 'From Matsuda et al. (1982). 'From Matsuda et al. (1986). 3Not measured.
0
0
0,2
0.4
0.6
0.8
1,O
ACROS I N
FIGURE 4 Correlation between the ratios of substrate specificity of acrosin and basic human seminal plasma arginine esterase (BHSAE).
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Human Acrosin
C
1
AMOUNT
3 OF
10
15
30
100
I N H I B I T O R ( pg )
FIGURE 5 Behavior of some proteinase inhibitors on human acrosin amidolytic activity. A mixture of the indicated amount of inhibitor and present acrosin (0.0012 A,,,) was preincubated at 30°C for 20 min and the remaining activity was measured by Val-Leu-Arg-pNA amidolytic activity. Activity is shown as a percentage of the untested control. 0,soybean trypsin inhibitor; 0,lima bean trypsin inhibitor; aprotinin; 0 , q-antitrypsin.
.,
detect any proacrosin. Our findings are also supported by the research by Anderson et al. [I] that frozen sperm contain no proacrosin. Human acrosin can be classified as a trypsin-like enzyme. There are many similarities between human acrosin and trypsin such as optimal pH (Fig. 2) and stimulation of enzyme activity by calcium ions (Fig. 3). However, the notable differences between the two enzymes are in their substrate specificity and their molecular mass. The human acrosin used by us has a similar specificity for Bz-Arg-Me to Tos-Arg-Me as shown in Table 2. This result is further supported by our previous report on boar acrosin 181; however, bovine pancreatic trypsin cleaves to Tos-Arg-Me about four times more rapidly than it cleaves to Bz-Arg-Me [9]. Moreover, the inhibitory spectrum of human acrosin in Fig. 5 differs from that of trypsin reported by Zaneveld et al. [16]. The substrate specificity and behavior in the presence of calcium ions of the acrosin in our study are clearly different from those of basic human seminal plasma arginine esterase [9], There is no basic proteinase inhibitor in seminal plasma in preparations that are not adsorbed on an LBTI affinity column (Fig. 1). This result indicates that the separation and purification of sperm from semen is complete, and also suggests that the acrosin in our study comes only from mature human sperm. We are currently studying the precise role of human acrosin during fertilization by using the purified enzyme described here.
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REFERENCES 1: Abderson RA, Berler SA, Mark SR, Zaneveld LDV (1981): Characterization of a high-molecular-weight form of human acrosin. Biochem J 199:307-316 2. Amudsen E, Putter J, Firberger P, Knos M, Larsbraten M, Claseson G (1979): Method for the determination of glandular kallikrein by means of a chromogenic substrate. In: Adv Exp Med Biol Vol. 120A, Kinin 11. Fujii S, Moriya H, and Suzuki T, (Eds.). New York: Plenum Press, pp 83-90 3. Baba T, Watanabe K, Kashiwabara S, Arai Y (1989): Primary structure of human proacrosin deduced from cDNA sequence. FEBS Letters 244:296-300 4. E k e JS, McIntyre J (1982): Purification of bovine and human acrosin. Can J Biochem 60:8-14 5. Ericsson RJ, Langevin CN, Nishino M (1973): Isolation of fractions rich in human Y sperm. Nature 246:421424 6. Kaneko S, Moriwaki C (1981): Studies on acrosin. 1. Purification and characterization of boar acrosin. J Pharm Dyn 4:20-27 7. Kaneko S, Oshio S , Kobanawa K, Kobayashi T, Mohri H, Iizuka R (1986): Purification of human sperm by a discontinuous Percoll density gradient with an inner column. Biol Reprod 35: 1059-1063 8. Matsuda Y, Miyazaki K, Fujimoto Y , Hojima Y, Moriya H (1982): Action of various kallikrein and related enzymes on synthetic arginine and lysine derivatives as substrate. Chem Pharm Bull 30: 1771-1775 9. Matsuda Y, Kaneko S, Oshio S, Iizuka R, Akihama S (1986): Basic arginine ester hydrolyzing enzymes in human urine and seminal plasma. Jap J Clin Chem 15:168-173 10. Moriwaki C, Hojima Y, Moriya H (1974): Use of combined assays of kallikrein activity measurement: a proposal. Chem Pharm Bull 22:975-981 11. Morton DB (1975): Acrosomal enzymes: immunochemical localization of acrosin and hyaluronidases in ram spermatozoa. J Reprod Fertil45:375-378 12. Schleuning WD, Schieeler H, Fritz H (1973): Highly purified acrosomal aproteinase (boar acrosin): isolation by affinity chromatography using benzamidine-cellulose and stabilization. Hoppe-Seyler’s Z. Physiol Chem 354:550-554 13. Siege1 MS, Bechtold DS, Kopta CI, Polakoski KL (1986): Characterization of human proacrosin using an automated fast protein liquid chromatography (FPLC) system. Biochim Biophys Acta 883:567-573 14. Stamgaugh R, Buckley J (1972): Studies on acrosomal proteinase of rabbit spermatozoa. Biochim Biophys Acta 284:473-477 15. Steeno 0, Adimoeja A, Steeno J (1975): Separation of X- and Y-bearing spermatozoa with Sephadex gel filtration method. Andrologia 7:95-97 16. Zaneveld LDJ, Polasoski KL, Williams WL (1972): Properties of proteolytic enzyme from rabbit sperm. Biol Reprod 6:30-39
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T. KOBAYASHI, Y. MATSUDA, S. OSHIO, S. KANEKO, S . NOZAWA, H. MHORI, S . AKIHAMA, and Y. FUJIMOTO
Human sperm with normal morphology and good viability were obtained by centrifugation using a discontinuous Percoll density gradient with an inner column. Acrosin (E.C.3.4.21.10) was rapidly purified from sperm by ion exchange adsorption and elution and was purified by affinity adsorption on a lima bean trypsin inhibitor (LBTI) Cellulofine column. The final preparation was found to be homogeneous on polyacrylamide gel electrophoresis and to have a molecular weight of about 4 x lo4 daltons. The enzyme had an esterolytic activity of 3.5 pnol/min/A280 with N-a-tosyl-L-arginine methyl ester as the substrate. Human acrosin showed a broad substrate specificity for arginine and lysine derivatives and it seemed to have a somewhat different specificity from trypsin. The optimal pH of this enzyme with amidolytic activity was 9.0. Enzyme activity was stimulated by a high concentration of calcium chloride. LBTI and aprotinin strongly suppressed the amidolytic activity with the o-valyl-Lleucyl-L-arginine-p-nitroanilide(Val-Leu-Arg-pNA) as the substrate, but cr,-antitrypsin and soybean trypsin inhibitor were less effective. Key Words: Acrosin; Human seminal plasma; Purification; Affinity chromatography; Substrate specificity; Arginine esterase.
INTRODUCTION Acrosin (E.C. 3.4.21.10) has been reported to be one of the trypsin-like serine proteinases associated with the acrosomal membrane of sperm. The functional role of acrosin in fertilization may be dissolution of the zona pellucida (1 1). Thus, the study of acrosin in human sperm is likely to yield important information on the mechanism of fertilization. Acrosin was first described in rabbit epididymal spermatozoa and mammalian acrosins were later purified by many investigators [4, 7, 12, 131. Human acrosins have been purified using washed sperm prepared by centrifugation of diluted semen. However, the washed sperm could not be freed from contamination with immature and abnormal sperm, other cellular
Received December 11, 1990. Accepted January 16, 1991. Department of Obstetrics and Gynecology, School of Medicine, Keio University (T.K., S.K., S.N.), Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. First Department of Biochemistry, Meiji College of Pharmacy (Y.M., S.A.), Nozawa, Setagaya-ku, Tokyo 154, Japan. Department of Biology, College of Arts and Sciences, University of Tokyo ( S . O . , H.M.), Komaba, Meguro-ku, Tokyo 153, Japan. Department of Clinical Biochemistry, Hokkaido Institute of Pharmaceutical Sciences (Y.F.), Otaru-Shi, Hokkaido 047-02, Japan. Address correspondence to: Dr. Y. Matsuda, First Department of Biochemistry, Meiji College of Pharmacy, Nozawa, Setagaya-ku, Tokyo, Japan. ARCHIVES OF ANDROLOGY 21:9-16 (1991) Copyright 0 1991 by Hemisphere Publishing Corporation
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components, and seminal plasma, so morphologically purified sperm preparations are required for accurate biochemical analysis and enzyme purification. Recently, we developed a Percoll density gradient centrifugation method using an inner column for the preparation of human sperm [7] that is capable of isolating motile sperm of normal morphology and decreasing seminal plasma to less than of that in original semen. In the present study we report the rapid purification of human acrosin and some of its properties using morphologically purified human sperm. Syst Biol Reprod Med Downloaded from informahealthcare.com by McMaster University on 11/14/14 For personal use only.
MATERIALSIMETHODS Purified Human Sperm Morphologically purificd human sperm were obtained using a discontinuous Percoll density gradient and inner-column centrifugation as described previously by Kaneko ct al. [7] Viable motile sperm in the sediment were resuspended in saline and werc frozen.
Enzyme Assay Esterolytic activity was colorirnetrically assayed using methyl ester derivatives as substrates and chromotropic acid at pH 8.0 and 30°C, as described by Moriwaki et al. [lo]. We used N-a-tosyl-Larginine methyl ester (Tos-Arg-Me) esterolytic activity to monitor the purification procedure of acrosin. Amidolytic activity was measured using D-valyl-L-leucyl-L-arginine-p-nitroanilide (Val-Leu-Arg-pNA, S-2266) as a substrate [2] at pH 8.0 and 30°C. The concentration of the esterolytic and amidolytic substrates were 10 and 0.5 mmol/L, respectively. All esterolytic and amidolytic activities were expressed in terms of pmol/L substrate hydrolyzed per minute (pmol/min) .
Protein Concentration The protein concentration was estimated spectrophotometrically assuming that the absorbance of 1mg protein in a 1-cm width cuvette at 280 nm is 1.O.
Reagents The following chemicals and proteins were obtained commercially: Aprotinin, CM-Cellulofine, Cellulofine GCL-300, and Formyl Cellulofine (Scikagaku Kogyo Company, Japan). Tos-Arg-Me, LBTI, bovine serum albumin (BSA), acetyl-glycyl-L-lysine methyl ester (Ac-Gly-Lys-Me), N-a-tosyl-L-lysine methyl ester (Tos-Lys-Me), acetyl-L-phenylalanine methyl ester (Ac-Phe-Me) and carbobenzoxy-Lalanyl-L-glycine methyl ester (CBA-Ala-Gly-Me) (Sigma Chemical Company, USA). N-a-Benzoyl-Larginine methyl ester (Bz-Arg-Me), acetyl-N-a-benzoyl-L-citrullinemethyl ester (Bz-Cit-Me) (Serva Chemical Company, Germany). a,-antitrypsin and soybean trypsin inhibitor (SBTI; Boehringer Mannheim, Germany).
RESULTS Samples of 18 ml of the purified sperm that contained lo9 cells/ml with more than 90% motility (normal morphology) were obtained from 100 ml human mixed semen. The purified human sperm were suspended in HCl solution at pH 2.0, containing 10%glycerin, and were crushed using Polytron (type 10/35). An equal volume of the same solution was added to the crushed sperm and shaken vigorously for 2 h. The extracted solution was centrifuged and the supernatant was dialyzed initially against deionized water for 6 h, and then against 0.01 mmol of phosphate buffer per liter at pH 7.0
11
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Human Acrosin
(buffer A) for 4 h. The dialyzed solution was applied to a CM-Cellulofine column preequilibrated with buffer A, and the column was washed with buffer A. The acrosin was eluted with buffer A containing 0.5 M NaCL, and the resulting solution was adjusted to pH 8.0. The active solution from the above step was applied to an LBTI Cellulofine column and the column was washed with 0.05 M Tris-HC1 buffer at pH 8.0 (buffer B). The first elution was made with buffer B containing 0.5 M CaCl and this preparation contained no Tos-Arg-Me esterolytic activity. The acrosin preparation that eluted with HC1 solution at pH 2.0 showed Tos-Arg-Me esterolytic activity (Fig. 1) and was adjusted to pH 8.0. The specific activity of this preparation was 3.50 and 0.161 pmol/min/A,,, for Tos-Arg-Me esterolytic activity and Val-Leu-Arg-pNA amidolytic activity, respectively. The overall results of the purification of human acrosin are summarized in Table 1. The specific activity was 51 times higher than that of the initial extract and the final preparation represented about 6.7 % recovery of Tos-Arg-Me esterolytic activity calculated from the initial extract. The isolated acrosin was homogeneous on polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 4 x lo4daltons by molecular sieve chromatography using a Cellulofine GCL-300 column. The Val-Leu-Arg-pNA amidolytic activity of acrosin, which was dependent upon pH, was measured using modified Britton-Robinson’s widerange buffer between pH 6.0 to 11.O, and the optimal pH was pH 9.0 (Fig. 2). The effect of calcium chloride on Val-Leu-Arg-pNA amidolytic activity of acrosin was tested (Fig. 3). Its effect on the amidolytic activity of basic human seminal plasma arginine esterase (BHSAE) is illustrated for comparison. Following the addition of 30- and 100-mM calcium chloride, the activity was observed to be 3.1 and 3.2 times higher than that of the control (with no addition of calcium chloride), respectively. However, a final concentration of
9
v
E 0 W
N
t-
a w
u z
a
m e
0
0
v)
m
CL IW -
4:
i n
w
0
10
FRACTION
20 NUMBER ( 2 , 5
u3
ml/tube
40 )
FIGURE 1 Lima bean trypsin inhibitor Cellulofine affinity adsorption of human acrosin. A, eluted with HCl solution at pH 2.0. 0 , absorbance at 280 nm;0, Tos-Arg-Me esterolytic activity.
T. Kobayashi et al.
12 TABLE 1 Purification of Human Acrosin
Esterolytic Activity
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Procedure Extract CM-cellulose adsorption and elution Lima bean trypsin inhibitor-Cellulofine affinity adsorption
Protein Recovery (%) 100
I .2 0.13
(%)
Specific Activity (pmol/min/A280)
100 13.1
0.068 0.124
Recovery
6.1
3.50
P.F. 1 1.8 51
P.F.. Purification factor.
more than 100 mM calcium chloride decreased the potentiation. Calcium chloride showed no potentiation effect on BHSAE. The esterolytic and amidolytic activities of enzymatically purified human acrosin on synthetic arginine, lysine, and other amino-acid derivatives used as substrates were examined (Table 2). Comparative data of substrate specificity or boar acrosin and BHSAE (Matsuda et al., 1982, 1986) are also shown in Table 2. Tos-Arg-Me and Bz-Arg-Me were easily hydrolyzed by present acrosin and the ratio of the activity of Tos-Arg-Me to Bz-Arg-Me was about 1. Acrosin did not attack Bz-Cit-Me or CBZ-Ala-Gly-Me. A correlation between the ratios of substrate specificity of Tos-Arg-Me to acrosin and BHSAE (Matsuda et al., 1986) was not observed (Fig. 4). Some proteinase inhibitors were tested on Val-Leu-Arg-pNA amidolytic activity of acrosin at pH 8.0 and 30" as shown in Fig. 5. Strong inhibition of acrosin was observed with LBTI and aprotinin whereas a,-antitrypsin and SBTI were less effective. The acrosin isolated in this study was not inhibited by fractions not adsorbed on an LBTI affinity adsorption column (fractions 3 to 10 in Figure 1).
DISCUSSION Human semen contains not only mature motile sperm, but also poor-quality sperm and other cellular components including bacteria. It is important to obtain morphologically normal, highly purified sperm preparations for the purification of an enzyme such as acrosin and for the accurate determination of its functional role in sperm because contaminants like bacteria are rich sources of enzymes and inhibitors. The isolation procedure used here facilitated the collection of normal, viable, and motile sperm without any contamination from seminal plasma or any other source; the purified acrosin was considered to be from mature sperm only. In the present study, human acrosin was purified from a sperm suspension containing lo9 cells/ ml of 90% motile cells. In addition, the present purification procedure is simpler and more rapid than that previously described by others [5, 151. The primary structure of human proacrosin was described by Baba et al. [3] using a deductive method with cDNA. The molecular mass of human proacrosin was predicted to be 43,860 d; the results of our study showed a similar molecular weight although we did not
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Human Acrosin
0’
13
’
6
8
7
9
10
11
PH FIGURE 2 pH dependence of amidolytic activity of human acrosin. The pH dependence of amidolytic activity of human acrosin was measured using Val-Leu-Arg-pNA as the substrate at various pH in 0.08 M modified Britton-Robinson’s wide range buffer. Activity is expressed as a percentage of that at optimal pH.
>-
-2 I-
3,O
I-
V
4 U
0
E: + a
2,o
CT
1,o
0
C
1
3
10
CONCENTRATION OF CaC1,
30 (
mM
100
300
1
FIGURE 3 Effects of calcium chloride on the esterolytic activity of human acrosin and basic human seminal plasma arginine esterase (BHSAE). Human acrosin or BHSAE and the indicated amount of calcium chloride were mixed and then Tos-Arg-Me esterolytic activity was measured. Activity is expressed as a ratio to the control (without calcium chloride). 0 , human acrosin; 0, BHSAE.
T. Kobayashi et al.
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TABLE 2 Esterolytic and Amidolytic Activities of Human Acrosin on Synthetic Arginine, Lysine, and Other Amino Acid Derivatives Used as Substrate
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Present Acrosin Substrate
Activity
Ratio
Tos-Arg-Me Bz-Arg-Me Ac-Phe- Arg-Me Ac-Lys-Me Tos-Lys-Me Ac-Gly-Lys-Me Bz-Cit-Me Ac-Phe-Me CBZ-Ala-Gly-Me Val-Leu- Arg-pNA
3.50 3.46 2.31 1.37 1.33 1.89 0 0 0 0.161
1 0.99 0.66 0.39 0.38 0.54 0 0 0 0.046
BHSAE' Ratio 1 0.20 0.73 0.63 1.09 0.94 0 0 0.02
Boar Acrosin' Ratio 1
1.01 3 3
0.43 1.56 3 3
3
3
3
The concentration of esterolytic and amidolytic substrate was 10 and 1 mM, respectively. All activities are expressed in pmol substrate hydrolyzed per min per A,,o at 30°C and pH 8.0. The ratio of activity is given relative to standard Tos-Arg-Me. 'From Matsuda et al. (1982). 'From Matsuda et al. (1986). 3Not measured.
0
0
0,2
0.4
0.6
0.8
1,O
ACROS I N
FIGURE 4 Correlation between the ratios of substrate specificity of acrosin and basic human seminal plasma arginine esterase (BHSAE).
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Human Acrosin
C
1
AMOUNT
3 OF
10
15
30
100
I N H I B I T O R ( pg )
FIGURE 5 Behavior of some proteinase inhibitors on human acrosin amidolytic activity. A mixture of the indicated amount of inhibitor and present acrosin (0.0012 A,,,) was preincubated at 30°C for 20 min and the remaining activity was measured by Val-Leu-Arg-pNA amidolytic activity. Activity is shown as a percentage of the untested control. 0,soybean trypsin inhibitor; 0,lima bean trypsin inhibitor; aprotinin; 0 , q-antitrypsin.
.,
detect any proacrosin. Our findings are also supported by the research by Anderson et al. [I] that frozen sperm contain no proacrosin. Human acrosin can be classified as a trypsin-like enzyme. There are many similarities between human acrosin and trypsin such as optimal pH (Fig. 2) and stimulation of enzyme activity by calcium ions (Fig. 3). However, the notable differences between the two enzymes are in their substrate specificity and their molecular mass. The human acrosin used by us has a similar specificity for Bz-Arg-Me to Tos-Arg-Me as shown in Table 2. This result is further supported by our previous report on boar acrosin 181; however, bovine pancreatic trypsin cleaves to Tos-Arg-Me about four times more rapidly than it cleaves to Bz-Arg-Me [9]. Moreover, the inhibitory spectrum of human acrosin in Fig. 5 differs from that of trypsin reported by Zaneveld et al. [16]. The substrate specificity and behavior in the presence of calcium ions of the acrosin in our study are clearly different from those of basic human seminal plasma arginine esterase [9], There is no basic proteinase inhibitor in seminal plasma in preparations that are not adsorbed on an LBTI affinity column (Fig. 1). This result indicates that the separation and purification of sperm from semen is complete, and also suggests that the acrosin in our study comes only from mature human sperm. We are currently studying the precise role of human acrosin during fertilization by using the purified enzyme described here.
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T. Kobayashi et al.
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REFERENCES 1: Abderson RA, Berler SA, Mark SR, Zaneveld LDV (1981): Characterization of a high-molecular-weight form of human acrosin. Biochem J 199:307-316 2. Amudsen E, Putter J, Firberger P, Knos M, Larsbraten M, Claseson G (1979): Method for the determination of glandular kallikrein by means of a chromogenic substrate. In: Adv Exp Med Biol Vol. 120A, Kinin 11. Fujii S, Moriya H, and Suzuki T, (Eds.). New York: Plenum Press, pp 83-90 3. Baba T, Watanabe K, Kashiwabara S, Arai Y (1989): Primary structure of human proacrosin deduced from cDNA sequence. FEBS Letters 244:296-300 4. E k e JS, McIntyre J (1982): Purification of bovine and human acrosin. Can J Biochem 60:8-14 5. Ericsson RJ, Langevin CN, Nishino M (1973): Isolation of fractions rich in human Y sperm. Nature 246:421424 6. Kaneko S, Moriwaki C (1981): Studies on acrosin. 1. Purification and characterization of boar acrosin. J Pharm Dyn 4:20-27 7. Kaneko S, Oshio S , Kobanawa K, Kobayashi T, Mohri H, Iizuka R (1986): Purification of human sperm by a discontinuous Percoll density gradient with an inner column. Biol Reprod 35: 1059-1063 8. Matsuda Y, Miyazaki K, Fujimoto Y , Hojima Y, Moriya H (1982): Action of various kallikrein and related enzymes on synthetic arginine and lysine derivatives as substrate. Chem Pharm Bull 30: 1771-1775 9. Matsuda Y, Kaneko S, Oshio S, Iizuka R, Akihama S (1986): Basic arginine ester hydrolyzing enzymes in human urine and seminal plasma. Jap J Clin Chem 15:168-173 10. Moriwaki C, Hojima Y, Moriya H (1974): Use of combined assays of kallikrein activity measurement: a proposal. Chem Pharm Bull 22:975-981 11. Morton DB (1975): Acrosomal enzymes: immunochemical localization of acrosin and hyaluronidases in ram spermatozoa. J Reprod Fertil45:375-378 12. Schleuning WD, Schieeler H, Fritz H (1973): Highly purified acrosomal aproteinase (boar acrosin): isolation by affinity chromatography using benzamidine-cellulose and stabilization. Hoppe-Seyler’s Z. Physiol Chem 354:550-554 13. Siege1 MS, Bechtold DS, Kopta CI, Polakoski KL (1986): Characterization of human proacrosin using an automated fast protein liquid chromatography (FPLC) system. Biochim Biophys Acta 883:567-573 14. Stamgaugh R, Buckley J (1972): Studies on acrosomal proteinase of rabbit spermatozoa. Biochim Biophys Acta 284:473-477 15. Steeno 0, Adimoeja A, Steeno J (1975): Separation of X- and Y-bearing spermatozoa with Sephadex gel filtration method. Andrologia 7:95-97 16. Zaneveld LDJ, Polasoski KL, Williams WL (1972): Properties of proteolytic enzyme from rabbit sperm. Biol Reprod 6:30-39
