Vol. 182, No. 2, 1992 January 31, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 667-674
TWO HIGH MOLECULAR MASS PROTEASES FROM SEA URCHIN SPERM Kazuo Inaba, Yasuhisa Akazome and Masaaki Morisawa Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Micra, Kanagawa 23&02, Japan Received
December
16,
1991
SUMMARY : Two-types of high molecular mass proteases have been purified from sea urchin sperm using DEAE-Sephacel, hydroxylapatite and Superdex 200 column chromatography. Both proteases showed similar hydrolyzing activities toward synthetic peptides, but they differed in the molecular mass and peptide composition. One was probably identical to a proteasome (multicatalytic proteinase) , judging from its molecular mass (650 kDa) and polypeptide composition. The other one was composed of several polypeptides with molecular masses ranging from 24 kDa to 12.5 kDa and its molecular mass was estimated as 950 kDa by gel filtration. These two proteases, however, were closely related to each other. Immunological studies revealed that the 950~kDa protease comprised at least five subunits of the 650~kDa protease. Q 1992Academic press, W.
Recently, several kinds of high molecular mass proteases have been isolated from many eukaryotic cells (1). In sperm cells, a multicatalytic proteinase, named proteasome (2), have been isolated from sea urchins and tunicates and possible participations in acrosome reaction have been discussed (3-S). In spite of many reports concerning high molecular mass proteases, the functions of them have been unclear. Since sperm is highly differentiated cell, it seems to be useful for investigating the functions and localizations of high molecular mass proteases. In the present study, we isolated two types of high molecular mass proteases from sea urchin sperm and examined the substrate specificities, susceptibilities to inhibitors and correlations between them. MATERIALS
AND METHODS
Preparation of High Molecular Mass Proteases Sperm from sea urchin, Anthocidaris crassispina, were obtained by the injection of 0.1 M acetylcholine into body cavity. Sperm Abbreviations : DTT, dithiothreitol ; MCA, 4-methylcoumaryl-7-amide ; HPLC, high performance liquid chromatography ; AMC, 7-amino-4-methylcoumarin ; SDS, sodium dodecyl sulfate ; PAGE, polyacrylamide gel electrophoresis ; TPCK, N-tosyl-phenylalanine chloromethyl ketone ; TLCK, N-tosyl-lysine chloromethyl ketone ; PMSF, phenylmethanesulfonyl fluoride ; 3,4DIC, 3,4dichloroisocoumarin ; NEM, N-ethylmaleimide ; CBBR, Coomassie Brilliant Blue R-250. 0006-291X/92 667
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
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No.
2, 1992
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AND
BIOPHYSICAL
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were washed with filtered sea water two or three times by centrifugation. Then, the pellet was suspended in three volumes of a extraction medium (0.1 % Triton X-100 (v/v), 0.15 M KCl, 2 mM MgCl,, 0.5 mM EGTA, 0.5 mM DTT, 10 mM Tris-HCl, pH 8.0) and stirred at 4°C for 1 hr, followed by the centrifugation at 15,000 rpm for 30 min (Kubota, RA-3 rotor). The obtained supematant (crude extract) was stored at -8O’C until used. The crude extract was centrifuged at 40,000 rpm for 1 hr (Hitachi, RP50 rotor). The obtained supematant was charged onto a Bio-Beads SM2 (Bio Rad) column (2 X 20 cm) to remove Triton X-100. The nonadsorbed fraction (670 mg total protein) was pooled and loaded on a DEAE-Sephacel (Pharmacia Fine Chemicals) colunm (2 X 20 cm) and then washed with T&on-free extraction medium. The adsorbed proteins were eluted with 0.15-0.6 M KC1 linear gradient. The fractions (eluted with 0.2-0.35 M KCl) which showed hydrolyzing activities toward Sue-Leu-Leu-Val-Tyr-MCA were pooled and dialyzed against 25 mM sodium phosphate buffer, pH 8.0, containing 2 mM MgCl,, 0.5 mM EGTA, 0.5 mM DTT. The retentate was charged onto a hydroxylapatite column (1.5 X 8 cm) and proteins were eluted with 25-250 mM sodium phosphate gradient. The peaks (around 175 mM sodium phosphate) of hydrolyzing activity toward Sue-Leu-Leu-Val-Tyr-MCA were pooled and finally loaded on HPLC Superdex 200 column (1.6 X 60 cm) (Pharmacia Fine Chemicals) and developed with T&on-free extraction medium at 1 ml / min. The active fractions were pooled and used for the experiments. Assay of Peptidase Activity The synthetic fluorescent peptides were dissolved into dimethylsulfoxide and 10 ~1 of the solution was added to a assay medium containing 0.15 M KCl, 2 mM MgCl,, 0.5 mM EGTA, 0.5 mM DTT, 10 mM Tris-HCl, pH 8.0. In the case of the examination of activation by SDS, KC1 was replaced with NaCl.The hydrolyzing activities toward synthetic peptides were measured by the fluorescence from released AMC using a fluorescence spectrophotometer (Hitachi, 650-10s) at the excitation and emission wavelength of 380 nm and 460 nm, respectively. SDS-PAGE, Immunoblotting and Protein Assay SDS-PAGE was performed by the method of Laemmli (6). For the preparation of rabbit antiserum against the 650-kDa protease, the CBBR-stained bands ranging from 22- to 33-kDa were used as antigen. Preparation of rabbit antiserum, affinity purification of antibody and immunoblotting were performed according to the method previously described (7). Protein concentration was determined by the method of Bradford (8) or by the absorbance at 280 nm for purified enzymes.
RESULTS The detergent-extract of the sea urchin sperm was subjected to DEAE-Sephacel column chromatography, followed by hydroxylapatite column chromatography. Through these two steps, the hydrolyzing activity toward Sue-Leu-Leu-Val-Tyr-MCA was recovered with a broad peak. However, the gel filtration with Superdex 200 column revealed that the active fraction contained two proteases(Fig. 1 ; fraction I and II). The re-HPLC of each peak with Superdex 200 showed clearly the existence of two high molecular mass proteases (Fig.2 and 3). The larger one (Fraction I) eluted between the void volume and the elution volume of thyroglobulin and the molecular mass was estimated as 950 kDa (Fig.2). SDS-PAGE showed that this enzyme was a complex that was composed of several polypeptides with molecular masses ranging from 24 kDa to 125 kDa. The molecular mass of smaller protease (peak II) was estimated as 650 kDa and was coeluted with at least ten kinds of polypeptides with molecular masses ranging from 22 kDa to 33 kDa (Fig.3). We then examined the substrate specificity of these two proteases (Table I). As far as ten kinds of synthetic substrates examined, these two proteases showed quite similar properties for substrate specificity, though the specific activity of the 950 kDa protease
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.0.
0.6 % is 2
0.6.
Fraction
Number
F&& Superdex 200 high performance gel filtration chromatography of the chymotrypsinlike active fraction obtained from hydroxylapatite column. Fractions of 1.3 ml were collected. The hydrolyzing activity toward Sue-L.eu-Leu-Val-Tyr-MCA of each fraction was expressed as “chymotrypsin-like activity”. The chymotrypsin-like activity was separated into two fractions (fraction I and 11).
was lower than that of the 650 kDa protease. Both proteases hydrolyzed not only a substrate for chymotrypsin-like proteases, but also the substrate for trypsin-like protease. The effects of several protease inhibitors on the hydrolyzing activities of two.proteases were also similar to each other but different in some points (Table II). The addition of two kinds of tosyl-chloromethane compounds gave little changes on the hydrolyzing activity of 950 kDa protease, whereas these two compounds acted as activators for the trypsin-like activity of 650 kDa protease. This was also the case with the effects of E-64 and pepstatin on both the chymotrypsin-like and the trypsin-like protease activities of the 950 kDa and 650 kDa proteases. Both chymotrypsin-like and trypsin-like activities of 950-kDa protease were inhibited by 3, QDIC, while the inhibition by this compound of the trypsin-like activity of 650 kDa protease was at the most 50 % of the control. The similarity in the substrate specificities and the susceptibility of inhibitors suggest the correlation between the two proteases. We then examined the existence of common components in these two proteases by using affinity-purified anti-65OkDa antibody (Fig.4). The result showed that the 950-kDa protease comprised at least five subunits ranging from 25 to 33-kDa of the 650-kDa protease, although CBBR-staining could not obviously detect these bands in the 950-kDa protease. Silver staining of the components of 950-kDa 669
Vol.
182, No. 2, 1992
BIOCHEMICAL
Fraction
AND BIOPHYSICAL
Number
RESEARCH COMMUNICATIONS
5
272829303l323334Js362728394641 +116
--20
F~J& Re-chromatography of fraction I on Superdex 200 HPLC column. A, elution pattern. The conditions of chromatography were the same as those in Fig. 1. Arrows indicate the column void volume (I), the elution volumes of thyroglobuhn (669 kDa) (2), catalase (245 kDa) (3), alcohol dehydrogenase (150 kDa) (4). bovine serum albumin (66 kDa) (5), ovalbumin (43 kDa) (6) , soybean trypsin inhibitor (20 kDa) (7), and the total bed volume of the column (8). The peak of chymotrypsin-like activity was in fraction 30. B, SDS-PAGE pattern (13 % gel) of the proteins of fraction 27 to fraction 41 (applied 10 ~1 of each fraction). The numbers at the right show the molecular masses of markers ; Pgalactosidase (116 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), lactate dehydrogenase (36.5 kDa), triosephosphate isomemse (26.6 kDa), soybean trypsin inhibitor
(20 kDa).
protease revealed the existence of 22- - 33-kDa polypeptides which showed the same electrophoretic mobilities as those in 650-kDa protease (Fig.4). The profiles of the activation of chymotrypsin-like activity caused by the addition of SDS significantly differed between these two proteases (Fig.5). SDS at 0.01 % activated the 650-kDa protease up to 11-fold of the activity in the absence of SDS, whereas the 950-kDa protease was activated up to only 2-fold. 670
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
.0.3.
T
o.2’
z : 0.1.
c
20
40 Fraction
60 Number
e.
80
z * 6
m
Re-chromatography of fraction II on Superdex 200 HPLC column. A, elution The peak of chymotrypsin-like activity was in fraction 33. B, SDS-PAGE pattern of the protein of fraction 26 to fraction 40 (applied 20~41of each fraction). The markers used for chromatography or SDS-PAGE were the same as those in Fig.%.
pattern.
DISCUSSION In the present study, we isolated two kinds of high molecular mass proteases from sea urchin sperm. These two enzymes showed similar substrate specificities and had not only chymotrypsin-like activity but also bypsin-like activity. The molecular masseswere, however, different and the susceptibility to inhibitors were somewhat different between them. Sperm proteases have been investigated with their possible involvements in acrosome reaction. The inhibition of acrosome reaction or fertilization by some protease inhibitors showed that chymotrypsin-like proteasesand/or trypsin-like proteases involve in acrosome reaction (9, 10). Multicatalytic proteinases (proteasomes) have been isolated from sea urchin and ascidian sperm as a chymotrypsin-like protease which may participate in 671
Vol.
182,
No.
2, 1992
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950 kDa attd 650 kDa proteases Activity(%) 950 kDa 650 kDa loo (0.72) 100 (3.7) 3 3 3 4
Sue-Leu-Lcu-Val-Tyr-MCA Sue-Ala-Pro-Ala-MCA Sue-Ala-Ala-ProPhc-MCA Boc-Lcu-Arg-Arg-MCA Boc-Gin-Ala-Arg-MCA Boc-Glu-Lys-Lys-MCA Sue-Gly-Pro-Lcu-Gly-Pro-MCA Arg-MCA Lcu-MCA Ala-MCA
292 15 0 0 0.03 0.04 0
500 14 0 0 0 0. 16 0
The activity toward Sue-Lcu-Lcu-Val-Tyr-MCA was dcfincd as 100 ‘70.The specific activity (mnolc/min/mg) oPcach cnzymc toward Sue-Lcu-Lcu-Val-Tyr-MCA is shown in parcnthcsis.
acrosome reaction (3-5). The 650 kDa protease identified in the present study is possibly the proteasome (2), judged by its high molecular mass, polypeptide composition and substrate specificity.
Table II. Effects qf various inhibitors otl the hydrolyzitrg activities toward Sue-LeuhuVal-Tyr-MCA (LLVY-MCA) and Boc-Lea-Arg-Arg-MCA (LRR-MCA) of both enzymes
Inhibitor
None TPCK TLCK Leupeptin Chymostatin Antipain PMSF E64 Pepstatin 3,4DIC NEM
Concentration (PM)
100 100 100 100 100 2000 10 10 100 1000
Activity (%) 950 kDa 650 kDa LLVY -MCA LRR-MCA LLVY -MCA LRR-MCA 100 85 85 83 5 57 70 94 79 8 8
100 78 87 3 3 9 75 91 94 9 4
672
100 93 94 37 1 77 39 147 225 8 2
100 298 159 7 1 2 73 148 203 52 4
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CBBR lmmunoblot 1
2
-
3
4
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ss -
5
P&& SDS-PAGE and anti-650-kDa immunoblot of the 9WkDa (lane 1 and 3) and the 650kDa (lane 2 and 4) proteases. After electrophoresis of the purified 950-kDa (6 pg) and 650-kDa (7pg) protease on 13 % gel, proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon, Millipore), which was used for CBBR- or immunostaining. Arrowheads indicate the common components recognized by anti-650-kDa antibody. Lnrre 5 shows the silver staining (X5’) pattern of the 950-kDa protease. Dots indicate the 32- - 33-kDa components which are present in the 950~kDa protease. P&Q. Activation of the chymotrypsin-like activities of the 950 (closed circle) and 650-kDa (open circle) proteases by SDS. The hydrolyzing activity toward Sue-LLVY-MCA of each enzyme in the absence of SDS was defined as 100 % and the relative activities were plotted against the concentration of SDS.
On the other hand, the 950 kDa protease have been lirst identified in sea urchin sperm in the present study. The immunoreactivity of the 25 w 33-kDa components of the 950-kDa protease with anti-650~kDa antibody and the profiles of SDS-stimulation of both proteases suggest that the 950-kDa protease is the “active” form of the 650~kDa protease. The active form of the eukaryotic 20 S proteasome has shown to sediment at 26 S (11, 12), which appeared to degrade ubiquitin-conjugated protein substrate in an ATPdependent manner (13, 14). Considering the significance of sperm-egg recognition at fertilization, it is possible that the 950 kDa protease also involves in acrosome reaction in ubiquitin-dependent manner. However, it should be noted that sperm proteases are not always derived from acrosome, since it is also possible that some proteases may be present in sperm flagella and involve in sperm motility (15, 16). The precise studies concerning the localizations of the 950 kDa and the 650 kDa proteases are required for the further discussion. ACKNOWLEDGMENTS This work was supported in part by grants-in-aid from the Japanese Ministry of Education, No.03740370 and No.03223206 to K.I. and No.01640003, No.01304006 and No.03404004 to M.M. 673
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REFERENCES 1. Rive& A.J. (1989) Biochem. J. 263,625-633. 2. Amigo, A.-P., Tanaka, K., Goldberg, A.L. and Welch, W.J. (1988) N&we, 331, 1!22194. 3. Ishii, S., Saitoh, Y. and Yokosawa, H. (1989) In Intracellular Proteolysis (Katsunuma, N. and Kominami, E., eds.), ~~225-232, Japan Sci. Sot. Press,Tokyo. 4. Saitoh, Y., Kawahara, H., Miyamatsu, H. and Yokosawa, H. (1991) Comp. B&hem. Phisiol. 99B, 71-76. 5. Matsumura, K. and Aketa, K. (1991) Mol. Reprod. Develop. 29, 189-199. 6. Laemmli, U.K. (1970) Nature 227,~685. 7. Inaba, K. and Mohri, H. (1989) J. Biochem. 106,349-354. 8. Bradford, M.M. (1976) Anal. Biochem. 72,248-252. 9. Green, J.D. and Summers, R.G. (1982) Dev. Biol. 93, 139-144. 10. Hoshi, M., Numakunai, T. and Sawada, H. (1981) Dev. Biol. 86, 117-121. 11. Armon, T., Ganoth, D. and Hershko, A. (1990) J. Biol. Chem. 265,2(X23-20726. 12. Orino, E., Tanaka, K., Tamum, T., Sone, S., Ogura, T. and Ichihara, A. (1991) FEBS Len. 284,206-210. 13. Waxman, L., Fagan, J.M. and Goldberg, A.L. (1987) J. Biol. Chem. %2,2451-2457. 14. Hough, R., Pratt, G. and Rechsteiner, M. (19%‘) J. Biol. Chem. 262,8303-83 13. 15. de Lamirande, E. and Gagnon, C. (19%) J. Cell Biol. 102, 1378-1383. 16. Inaba, K. and Morisawa, M. (1991) Biomed. Res. 12,435-437.
674
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 667-674
TWO HIGH MOLECULAR MASS PROTEASES FROM SEA URCHIN SPERM Kazuo Inaba, Yasuhisa Akazome and Masaaki Morisawa Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Micra, Kanagawa 23&02, Japan Received
December
16,
1991
SUMMARY : Two-types of high molecular mass proteases have been purified from sea urchin sperm using DEAE-Sephacel, hydroxylapatite and Superdex 200 column chromatography. Both proteases showed similar hydrolyzing activities toward synthetic peptides, but they differed in the molecular mass and peptide composition. One was probably identical to a proteasome (multicatalytic proteinase) , judging from its molecular mass (650 kDa) and polypeptide composition. The other one was composed of several polypeptides with molecular masses ranging from 24 kDa to 12.5 kDa and its molecular mass was estimated as 950 kDa by gel filtration. These two proteases, however, were closely related to each other. Immunological studies revealed that the 950~kDa protease comprised at least five subunits of the 650~kDa protease. Q 1992Academic press, W.
Recently, several kinds of high molecular mass proteases have been isolated from many eukaryotic cells (1). In sperm cells, a multicatalytic proteinase, named proteasome (2), have been isolated from sea urchins and tunicates and possible participations in acrosome reaction have been discussed (3-S). In spite of many reports concerning high molecular mass proteases, the functions of them have been unclear. Since sperm is highly differentiated cell, it seems to be useful for investigating the functions and localizations of high molecular mass proteases. In the present study, we isolated two types of high molecular mass proteases from sea urchin sperm and examined the substrate specificities, susceptibilities to inhibitors and correlations between them. MATERIALS
AND METHODS
Preparation of High Molecular Mass Proteases Sperm from sea urchin, Anthocidaris crassispina, were obtained by the injection of 0.1 M acetylcholine into body cavity. Sperm Abbreviations : DTT, dithiothreitol ; MCA, 4-methylcoumaryl-7-amide ; HPLC, high performance liquid chromatography ; AMC, 7-amino-4-methylcoumarin ; SDS, sodium dodecyl sulfate ; PAGE, polyacrylamide gel electrophoresis ; TPCK, N-tosyl-phenylalanine chloromethyl ketone ; TLCK, N-tosyl-lysine chloromethyl ketone ; PMSF, phenylmethanesulfonyl fluoride ; 3,4DIC, 3,4dichloroisocoumarin ; NEM, N-ethylmaleimide ; CBBR, Coomassie Brilliant Blue R-250. 0006-291X/92 667
$1.50
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
182,
No.
2, 1992
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AND
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RESEARCH
COMMUNICATIONS
were washed with filtered sea water two or three times by centrifugation. Then, the pellet was suspended in three volumes of a extraction medium (0.1 % Triton X-100 (v/v), 0.15 M KCl, 2 mM MgCl,, 0.5 mM EGTA, 0.5 mM DTT, 10 mM Tris-HCl, pH 8.0) and stirred at 4°C for 1 hr, followed by the centrifugation at 15,000 rpm for 30 min (Kubota, RA-3 rotor). The obtained supematant (crude extract) was stored at -8O’C until used. The crude extract was centrifuged at 40,000 rpm for 1 hr (Hitachi, RP50 rotor). The obtained supematant was charged onto a Bio-Beads SM2 (Bio Rad) column (2 X 20 cm) to remove Triton X-100. The nonadsorbed fraction (670 mg total protein) was pooled and loaded on a DEAE-Sephacel (Pharmacia Fine Chemicals) colunm (2 X 20 cm) and then washed with T&on-free extraction medium. The adsorbed proteins were eluted with 0.15-0.6 M KC1 linear gradient. The fractions (eluted with 0.2-0.35 M KCl) which showed hydrolyzing activities toward Sue-Leu-Leu-Val-Tyr-MCA were pooled and dialyzed against 25 mM sodium phosphate buffer, pH 8.0, containing 2 mM MgCl,, 0.5 mM EGTA, 0.5 mM DTT. The retentate was charged onto a hydroxylapatite column (1.5 X 8 cm) and proteins were eluted with 25-250 mM sodium phosphate gradient. The peaks (around 175 mM sodium phosphate) of hydrolyzing activity toward Sue-Leu-Leu-Val-Tyr-MCA were pooled and finally loaded on HPLC Superdex 200 column (1.6 X 60 cm) (Pharmacia Fine Chemicals) and developed with T&on-free extraction medium at 1 ml / min. The active fractions were pooled and used for the experiments. Assay of Peptidase Activity The synthetic fluorescent peptides were dissolved into dimethylsulfoxide and 10 ~1 of the solution was added to a assay medium containing 0.15 M KCl, 2 mM MgCl,, 0.5 mM EGTA, 0.5 mM DTT, 10 mM Tris-HCl, pH 8.0. In the case of the examination of activation by SDS, KC1 was replaced with NaCl.The hydrolyzing activities toward synthetic peptides were measured by the fluorescence from released AMC using a fluorescence spectrophotometer (Hitachi, 650-10s) at the excitation and emission wavelength of 380 nm and 460 nm, respectively. SDS-PAGE, Immunoblotting and Protein Assay SDS-PAGE was performed by the method of Laemmli (6). For the preparation of rabbit antiserum against the 650-kDa protease, the CBBR-stained bands ranging from 22- to 33-kDa were used as antigen. Preparation of rabbit antiserum, affinity purification of antibody and immunoblotting were performed according to the method previously described (7). Protein concentration was determined by the method of Bradford (8) or by the absorbance at 280 nm for purified enzymes.
RESULTS The detergent-extract of the sea urchin sperm was subjected to DEAE-Sephacel column chromatography, followed by hydroxylapatite column chromatography. Through these two steps, the hydrolyzing activity toward Sue-Leu-Leu-Val-Tyr-MCA was recovered with a broad peak. However, the gel filtration with Superdex 200 column revealed that the active fraction contained two proteases(Fig. 1 ; fraction I and II). The re-HPLC of each peak with Superdex 200 showed clearly the existence of two high molecular mass proteases (Fig.2 and 3). The larger one (Fraction I) eluted between the void volume and the elution volume of thyroglobulin and the molecular mass was estimated as 950 kDa (Fig.2). SDS-PAGE showed that this enzyme was a complex that was composed of several polypeptides with molecular masses ranging from 24 kDa to 125 kDa. The molecular mass of smaller protease (peak II) was estimated as 650 kDa and was coeluted with at least ten kinds of polypeptides with molecular masses ranging from 22 kDa to 33 kDa (Fig.3). We then examined the substrate specificity of these two proteases (Table I). As far as ten kinds of synthetic substrates examined, these two proteases showed quite similar properties for substrate specificity, though the specific activity of the 950 kDa protease
Vol.
182, No. 2, 1992
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
1.0.
0.6 % is 2
0.6.
Fraction
Number
F&& Superdex 200 high performance gel filtration chromatography of the chymotrypsinlike active fraction obtained from hydroxylapatite column. Fractions of 1.3 ml were collected. The hydrolyzing activity toward Sue-L.eu-Leu-Val-Tyr-MCA of each fraction was expressed as “chymotrypsin-like activity”. The chymotrypsin-like activity was separated into two fractions (fraction I and 11).
was lower than that of the 650 kDa protease. Both proteases hydrolyzed not only a substrate for chymotrypsin-like proteases, but also the substrate for trypsin-like protease. The effects of several protease inhibitors on the hydrolyzing activities of two.proteases were also similar to each other but different in some points (Table II). The addition of two kinds of tosyl-chloromethane compounds gave little changes on the hydrolyzing activity of 950 kDa protease, whereas these two compounds acted as activators for the trypsin-like activity of 650 kDa protease. This was also the case with the effects of E-64 and pepstatin on both the chymotrypsin-like and the trypsin-like protease activities of the 950 kDa and 650 kDa proteases. Both chymotrypsin-like and trypsin-like activities of 950-kDa protease were inhibited by 3, QDIC, while the inhibition by this compound of the trypsin-like activity of 650 kDa protease was at the most 50 % of the control. The similarity in the substrate specificities and the susceptibility of inhibitors suggest the correlation between the two proteases. We then examined the existence of common components in these two proteases by using affinity-purified anti-65OkDa antibody (Fig.4). The result showed that the 950-kDa protease comprised at least five subunits ranging from 25 to 33-kDa of the 650-kDa protease, although CBBR-staining could not obviously detect these bands in the 950-kDa protease. Silver staining of the components of 950-kDa 669
Vol.
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BIOCHEMICAL
Fraction
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Number
RESEARCH COMMUNICATIONS
5
272829303l323334Js362728394641 +116
--20
F~J& Re-chromatography of fraction I on Superdex 200 HPLC column. A, elution pattern. The conditions of chromatography were the same as those in Fig. 1. Arrows indicate the column void volume (I), the elution volumes of thyroglobuhn (669 kDa) (2), catalase (245 kDa) (3), alcohol dehydrogenase (150 kDa) (4). bovine serum albumin (66 kDa) (5), ovalbumin (43 kDa) (6) , soybean trypsin inhibitor (20 kDa) (7), and the total bed volume of the column (8). The peak of chymotrypsin-like activity was in fraction 30. B, SDS-PAGE pattern (13 % gel) of the proteins of fraction 27 to fraction 41 (applied 10 ~1 of each fraction). The numbers at the right show the molecular masses of markers ; Pgalactosidase (116 kDa), bovine serum albumin (68 kDa), ovalbumin (45 kDa), lactate dehydrogenase (36.5 kDa), triosephosphate isomemse (26.6 kDa), soybean trypsin inhibitor
(20 kDa).
protease revealed the existence of 22- - 33-kDa polypeptides which showed the same electrophoretic mobilities as those in 650-kDa protease (Fig.4). The profiles of the activation of chymotrypsin-like activity caused by the addition of SDS significantly differed between these two proteases (Fig.5). SDS at 0.01 % activated the 650-kDa protease up to 11-fold of the activity in the absence of SDS, whereas the 950-kDa protease was activated up to only 2-fold. 670
Vol.
182, No. 2, 1992
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
.0.3.
T
o.2’
z : 0.1.
c
20
40 Fraction
60 Number
e.
80
z * 6
m
Re-chromatography of fraction II on Superdex 200 HPLC column. A, elution The peak of chymotrypsin-like activity was in fraction 33. B, SDS-PAGE pattern of the protein of fraction 26 to fraction 40 (applied 20~41of each fraction). The markers used for chromatography or SDS-PAGE were the same as those in Fig.%.
pattern.
DISCUSSION In the present study, we isolated two kinds of high molecular mass proteases from sea urchin sperm. These two enzymes showed similar substrate specificities and had not only chymotrypsin-like activity but also bypsin-like activity. The molecular masseswere, however, different and the susceptibility to inhibitors were somewhat different between them. Sperm proteases have been investigated with their possible involvements in acrosome reaction. The inhibition of acrosome reaction or fertilization by some protease inhibitors showed that chymotrypsin-like proteasesand/or trypsin-like proteases involve in acrosome reaction (9, 10). Multicatalytic proteinases (proteasomes) have been isolated from sea urchin and ascidian sperm as a chymotrypsin-like protease which may participate in 671
Vol.
182,
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Able
BIOCHEMICAL
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qf the
1. Sdxtmte
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950 kDa attd 650 kDa proteases Activity(%) 950 kDa 650 kDa loo (0.72) 100 (3.7) 3 3 3 4
Sue-Leu-Lcu-Val-Tyr-MCA Sue-Ala-Pro-Ala-MCA Sue-Ala-Ala-ProPhc-MCA Boc-Lcu-Arg-Arg-MCA Boc-Gin-Ala-Arg-MCA Boc-Glu-Lys-Lys-MCA Sue-Gly-Pro-Lcu-Gly-Pro-MCA Arg-MCA Lcu-MCA Ala-MCA
292 15 0 0 0.03 0.04 0
500 14 0 0 0 0. 16 0
The activity toward Sue-Lcu-Lcu-Val-Tyr-MCA was dcfincd as 100 ‘70.The specific activity (mnolc/min/mg) oPcach cnzymc toward Sue-Lcu-Lcu-Val-Tyr-MCA is shown in parcnthcsis.
acrosome reaction (3-5). The 650 kDa protease identified in the present study is possibly the proteasome (2), judged by its high molecular mass, polypeptide composition and substrate specificity.
Table II. Effects qf various inhibitors otl the hydrolyzitrg activities toward Sue-LeuhuVal-Tyr-MCA (LLVY-MCA) and Boc-Lea-Arg-Arg-MCA (LRR-MCA) of both enzymes
Inhibitor
None TPCK TLCK Leupeptin Chymostatin Antipain PMSF E64 Pepstatin 3,4DIC NEM
Concentration (PM)
100 100 100 100 100 2000 10 10 100 1000
Activity (%) 950 kDa 650 kDa LLVY -MCA LRR-MCA LLVY -MCA LRR-MCA 100 85 85 83 5 57 70 94 79 8 8
100 78 87 3 3 9 75 91 94 9 4
672
100 93 94 37 1 77 39 147 225 8 2
100 298 159 7 1 2 73 148 203 52 4
Vol.
182, No. 2, 1992
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CBBR lmmunoblot 1
2
-
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ss -
5
P&& SDS-PAGE and anti-650-kDa immunoblot of the 9WkDa (lane 1 and 3) and the 650kDa (lane 2 and 4) proteases. After electrophoresis of the purified 950-kDa (6 pg) and 650-kDa (7pg) protease on 13 % gel, proteins were transferred onto a polyvinylidene difluoride membrane (Immobilon, Millipore), which was used for CBBR- or immunostaining. Arrowheads indicate the common components recognized by anti-650-kDa antibody. Lnrre 5 shows the silver staining (X5’) pattern of the 950-kDa protease. Dots indicate the 32- - 33-kDa components which are present in the 950~kDa protease. P&Q. Activation of the chymotrypsin-like activities of the 950 (closed circle) and 650-kDa (open circle) proteases by SDS. The hydrolyzing activity toward Sue-LLVY-MCA of each enzyme in the absence of SDS was defined as 100 % and the relative activities were plotted against the concentration of SDS.
On the other hand, the 950 kDa protease have been lirst identified in sea urchin sperm in the present study. The immunoreactivity of the 25 w 33-kDa components of the 950-kDa protease with anti-650~kDa antibody and the profiles of SDS-stimulation of both proteases suggest that the 950-kDa protease is the “active” form of the 650~kDa protease. The active form of the eukaryotic 20 S proteasome has shown to sediment at 26 S (11, 12), which appeared to degrade ubiquitin-conjugated protein substrate in an ATPdependent manner (13, 14). Considering the significance of sperm-egg recognition at fertilization, it is possible that the 950 kDa protease also involves in acrosome reaction in ubiquitin-dependent manner. However, it should be noted that sperm proteases are not always derived from acrosome, since it is also possible that some proteases may be present in sperm flagella and involve in sperm motility (15, 16). The precise studies concerning the localizations of the 950 kDa and the 650 kDa proteases are required for the further discussion. ACKNOWLEDGMENTS This work was supported in part by grants-in-aid from the Japanese Ministry of Education, No.03740370 and No.03223206 to K.I. and No.01640003, No.01304006 and No.03404004 to M.M. 673
Vol.
182,
No.
2, 1992
BIOCHEMICAL
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BIOPHYSICAL
RESEARCH
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