purification of an acrosin-like enzyme from sea urchin sperm*

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 255. No. 10. Issue of May 25, pp. 4814420 , 1980 Printed in U.S.A.

Purification of an Acrosin-like Enzyme from Sea Urchin Sperm*

(Received for publication, September 24, 1979)

Alan E. Levine$ and Kenneth A. Walsh From the Department of Biochemistry, University of Washington, Seattle, Washington 98195

Sea urchin sperm contain an acrosin-like enzyme with an apparent molecular weight of 53,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis re- veals two subunits of 34,000 daltons and 18,000 daltons. The M, = 34,000 subunit is the catalytic entity as revealed both by labeling with [14C]diisopropyl phosphorofluoridate and by proteolytic activity after dissociation of the subunits at pH 2.5. Both the 34,000-dalton and the 53,000-dalton forms of the enzyme catalyze the hydrolysis of W-benzoyl-L-arginine ethyl ester and both are inactivated by inhibitors of low molecular weight, whereas only the M, = 34,000 form is inactivated by large proteinaceous inhibitors. Only the M, = 34,000 form catalyzes proteolysis of denatured lysozyme or the B chain of insulin. The M, = 18,000 subunit appears to suppress the proteolytic activity but not the activity toward the small ester substrate. These findings are discussed in terms of possible roles of this enzyme in the control of early events leading to fertilization.

T h e eggs of most species are surrounded by a variety of investments which sperm must penetrate in the fertilization process. Once this is accomplished, the plasma membranes of the two gametes can fuse to form the zygote. The acrosomal granule, found in the anterior region of the sperm, is thought to contain the enzymes necessary for this penetration (re- viewed in Refs. 1 and 2), as well as components involved in specific recognition (3) and binding of sperm to the egg (4). These components are exposed when the sperm undergoes the acrosome reaction.

A proteolytic enzyme, acrosin, has been isolated from several species of vertebrate sperm (2,5-8) and is reported to aid in penetration of the zona pellucida (9). Acrosin has many properties in common with vertebrate trypsin (2, 10, ll), including its occurrence as a zymogen, proacrosin, found in both testes and sperm (12-15). Several lytic activities have also been associated with marine invertebrate sperm (l), but none has been identified as acrosin and none has been found to occur as a zymogen.

We have previously demonstrated an acrosin-like enzyme in the sperm of the sea urchin Strongylocentrotuspurpuratus (16). This enzyme is masked in freshly shed sperm but can be exposed to exogenous substrate and inhibitors by treatment of sperm with either 30 mM CaC12 or low concentrations of

* This work was supported by Grant GM-15731 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

+Supported by a National Research Service Award from the National Institutes of Health (GM 07270). Present address, Depart- ment of Neurobiology, Stanford University, School of Medicine, Stan- ford. CA 94305.

egg jelly coat. Inhibition of the enzyme prevents fertilization, apparently by blocking filament extrusion in the acrosome reaction (16, 17). In this report, we describe the purification and characterization of this protease from sea urchin sperm and from testes.

MATERIALS AND METHODS

[3H]BzArgOEt' was synthesized as previously described (16). [14C]DFP, [3H]DFP, ['4C]iodoacetamide, Liquifluor, Protosol, Aqua- sol, and Omnifluor were purchased from New England Nuclear.

Biological Materials-Sea urchin (S. purpuratus) gametes were obtained as preyiously described (16). Sperm were washed by suspen- sion in calcium-free artificial seawater and centrifuged at 1000 X g for 10 min. Sperm concentrations were estimated by the absorbance of suspensions at 340 nm, using 1.0 cm" for 1.2 X 10' sperm/ml (18). Sperm were used directly or frozen at -20°C. Frozen material was thawed at 4°C before homogenization.

Artificial seawater and egg jelly coat were prepared as described previously (16). The acrosome reaction was triggered by the addition of jelly coat preparations to give a final concentration of 4 pg of fucose/ml. Filament extension was examined by phase contrast mi- croscopy (17).

Homogenization of Tissue-Testes or sperm were homogenized in 3 volumes of cold buffer in a Virtis 45 homogenizer at low speed for 2 min. The homogenates were stirred overnight at 4OC and centrifuged at 30,000 X g for 1 h. The supernatant was then filtered through Whatman No. 1 filter paper, dialyzed against 1 mM HC1 at 4'C overnight, and centrifuged as above. In some experiments, sperm were disrupted using a Dounce apparatus.

Enzyme from Acrosome-reacted Sperm-Sperm (10 m l ) were sus- pended in artificial seawater containing 5 mM Tris, pH 8 (800 ml), and the acrosome reaction induced by the addition of egg jelly coat (17). After 15 min, the sperm were removed by centrifugation and the supernatant was concentrated to 29 ml using an Amicon apparatus with a UM-10 membrane.

Assay of Esterase Activity-The hydrolysis of r3H]BzArgOEt (labeled with tritium in the ethanol moiety) was followed by the method previously described (16) as modified for continuous monitoring of released tritiated ethanol (19). The aqueous solution contained enzyme, 0.13 M Tris, pH 8, and 1.25 mM [3H]BzArgOEt. One unit of activity is defined as the amount of enzyme catalyzing the hydrolytic release of 10 cpm/min, which corresponds to 180 pmol of substrate hydrolyzed/min. The assay was carried out in a Packard Tri-Carb scintillation counter cooled to 12OC. In experiments with inhibitors, the enzyme was preincubated with the inhibitor at room temperature (pH 8.0) for 20 min, then substrate was added and hydrolysis was measured as described above. The pH optimum was determined using 0.02 M acetate/Mes/Hepes between pH 4 and 7.5, and using 0.02 M Hepes/Tris between pH 7.5 and 9.

Assay of Protease Activity-Proteolysis was estimated by the release of trichloroacetic acid-soluble radioactivity from ['4C]carbox- amidomethyl lysozyme (5 pCi/mmol) prepared essentially by the method of Crestfield et al. (20). Lysozyme (100 mg) was dissolved in

' The abbreviations used are: BzArgOEt, N"-benzoyl-L-arginine ethyl ester; DFP, diisopropyl phosphorofluoridate; PMSF, phenyl- methanesulfonyl fluoride; SDS, sodium dodecyl sulfate; STI, soybean trypsin inhibitor (Kunitz); Mes, 2-(N-morpholino)ethanesulfonic acid Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Pipes, 1,4-piperazinediethanesulfonic acid. 18,000 and 34,OOO refer to the 18,000-dalton and 34,000-dalton subunits of the 53,000-dalton enzyme (53,Ow.

4814

Sea Urchin Acrosin 4815

10 ml of a buffer containing 6 M guanidine HCI, 50 mM EDTA, 0.5 M Tris at pH 8.6. A 100-fold molar excess of dithiothreitol was added, the mixture was incubated under Nz for 2 h at room temperature, then 100 pCi (13 mg) of [14C]iodoacetamide was added in 0.5 ml of 0.2 N NaOH. After 20 min, the alkylation was completed by the addition of 1 g of unlabeled iodoacetamide. After 75 min, the reaction mixture was adjusted to pH 6.5 and dialyzed against several changes of distilled water, then 0.01 N HCl, and stored at -2OOC. Some experiments utilized a more highly labeled derivative ([14C]carboxymethyl lysozyme) which was a gift from Dr. D. Pannelee. It had a specific activity of 20 mCi/mmol.

Each assay contained ['4C]lysozyme (0.85 mg/ml), 0.2 M Tris (pH 8), and enzyme. After various times at room temperature, an aliquot was treated with an equal volume of cold 10% trichloroacetic acid for 15 min, then centrifuged in an Eppendorf 3200 centrifuge (12,000 rpm). An aliquot of the supernatant was added to 10 ml of Aquasol, and the amount of radioactivity was determined. For experiments with inhibitors, the enzyme and inhibitor were incubated for 20 min at room temperature before addition of substrate.

Digestion of Oxidized B Chain of Insulin-The 34,000-dalton species of enzyme was dialyzed against 20 mM ammonium acetate, pH 5, and 0.23 unit was added to 50 nmol of oxidized B chain of bovine insulin in 155 pl of 1% NH4HC03, pH 8, incubated overnight at room temperature and lyophilized. A drop of water was added and the solution applied to Whatman No. 1 paper. High voltage electrophoresis was performed (40 V/cm) at pH 6.5 for 90 min (21). A guide strip, also containing standard amino acids, was stained with ninhy- drin.

Gel Electrophoresis-SDS-polyacrylamide slab gel electrophoresis was performed according to the method of Studier (22) using 5% acrylamide in the stacking gel and 15% acrylamide in the separating gel. Protein samples were boiled for 3 min in an incubation buffer containing 1% SDS, 1% P-mercaptoethanol, pH 6.8. Standard proteins used were bovine serum albumin (M, = 67,000), ovalbumin (M, = 45,000), myoglobin (M, = 17,000), and lysozyme (M, = 14,000). Gels containing radioactive proteins were cut into 2.4-mm slices and each slice was incubated overnight at 40°C in 1 ml of Protosol containing 1 drop of water. After cooling, 10 ml of 4% Omnifluor in toluene was added, and the samples were counted.

Labeling of Enzyme with [14C]DFP-Enzyme (3 to 50 units in 2.5 ml, pH 8) was treated with 100 pl of [I4C]DFP (103 mCi/mmol, 2 mM in propylene glycol) at room temperature. After 1 h, the labeled enzyme was separated from excess DFP on a small column of Seph- adex G-25 in 0.1 M formic acid or by dialysis. For measurements of the specific enzyme activity, radiolabeled DFP was diluted to 0.5 M with a solution of unlabeled DFP in isopropyl alcohol. The specific radioactivity of this labeled DFP solution was measured according to the method of Robinson et al. (23).

lution (2 units in 0.5 ml) was adjusted to pH 6 and applied (10 ml/h) Chromatography on Concanavalin A-Sepharose-An enzyme so-

to a small column (0.5 x l cm) of concanavalin A-Sepharose (Phar- macia) equilibrated with 0.5 M NaC1,0.05 M Pipes, pH 6. After washing the column with 5 ml of the starting buffer, glycoproteins were eluted with 0.2 M a-methylmannoside, 1 M NaCI, 0.05 M Pipes, pH 6.

STZ-Sepharose-Sepharose was activated with CNBr and €-ami- nohexanoic acid was coupled to it as described by Anderson et al. (19). Soybean trypsin inhibitor (STI) was attached by adding l-ethyl- 3-(3-dimethylaminopropyl) carbodiimide (6.7 mg/ml in 0.1 M Mes, pH 6) dropwise to 10 ml of wet, packed e-aminohexanoyl-Sepharose in 10 ml of Mes buffer, pH 6. After 3 min, ST1 (5 mg/ml, pH 6 ) was added dropwise, maintaining the pH at 6. After overnight reaction, the product was thoroughly washed. This product bound 1.2 mg of trypsin/ml of gel.

Cross-linking of Enzyme with Dimethyl Suberimidate-The method of Davies and Stark (24) was used. Enzyme (42 units/ml, final concentration), labeled with [I4C]DFP, was incubated in 0.17 M tri- ethanolamine (pH 8.5) containing dimethyl suberimidate (6 mg/ml) for 90 min at room temperature. The reaction mixture, and a control lacking the suberimidate, were dialyzed against distilled water, lyophilized, and examined by SDS-polyacrylamide slab gel electrophoresis.

LKB flatbed electrofocusing apparatus (model 2117, Multiphor) es- Isoelectric Focusing-Isoelectric focusing was performed using an

sentially as described in LKB Application Note 198. An ampholine range of pH 3 to 6 was used with an Ultrodex (LKB) supporting gel. A testes extract (1250 units in 243 m l ) was dialyzed against 1 mM HCl, concentrated to 20 ml in an Amicon ultrafiltration apparatus using a UM-10 membrane, and applied in the gel slurry to the supporting plate. Excess water was evaporated and the voltage applied (initially

730 V) for 14% h at 1-2°C. A grid was then placed in the gel bed to prevent diffusion. A small amount of the gel was removed from each fraction and mixed with 1 ml of distilled water, and the pH was measured. To elute the protein, the remaining gel was transferred from the grid to a 5-cm3 syringe fitted with a glass wool plug, and irrigated with 7 ml of 0.05 M sodium formate, 0.5 M NaCI, pH 3. Each eluate was assayed for enzymatic activity toward ["HIBzArgOEt.

RESULTS

Selection of Extraction Conditions

Since acrosin has been shown to exist as a zymogen in the sperm and testes of several vertebrate species (12-15), we sought evidence of a zymogen in sea urchin sperm after various extraction procedures (Table I). The observed amount of enzyme activity is little changed by varying the pH and divalent ion concentration or by including benzamidine (a competitive inhibitor of trypsin-like enzymes). Therefore, either the acrosin-like enzyme exists as an active species in sea urchin sperm (and testes), or appropriate conditions to prevent activation of the putative zymogen were not found. Since the enzyme is stable in acid, most of the subsequent purification steps were carried out at pH 3 (4°C) to preclude autolysis.

Isolation from Testes Since a larger quantity of the enzyme can be obtained from

sea urchin testes than from sperm, initial efforts used this tissue. Testes were homogenized in 0.1 M ammonium formate, pH 3, and an extract prepared as described under "Materials and Methods."

Solid NaCl was added to the extract to a final concentration of 0.15 M and the solution was applied to an SP-Sephadex C- 25 column at pH 3 (Fig. 1). All enzymatic activity was retained on this column and eluted at approximately 0.25 M NaCl with a yield of 55%. The active fractions were pooled and stored at 4°C. The isoelectric point of the active component was determined to be 4.5.

Molecular Weight Determinations

SDS-polyacrylamide gel electrophoresis of the active fractions pooled in Fig. 1, showed two major proteins of M, = 34,000 and 18,000 which stain with approximately the same intensity (Fig. 1, inset). Inclusion of a reducing agent during sample preparation did not alter the apparent molecular weight of these two bands. When the enzyme was labeled with [I4C]DFP before electrophoresis, the radioactivity was asso-

TABLE I Effect of extraction conditions on esterase activity

Sperm

Homogenization conditions BzArgOEt hydrolysis (units/sperm) X IO"

0.5 M NaCI, pH 2.5 3.0 0.5 M NaCI, pH 4.0 3.4 0.5 M NaCI, pH 7.0 3.6 0.5 M NaCl, pH 4.0, 25 m~ CaCb 2.8 0.5 M NaCI, pH 4.0, 10 mM EGTA" 3.5 0.5 M NaCI, pH 4.0, 1 m~ o-phenanthroline 3.8 0.5 M NaCI, pH 4.0, 10 mM N-ethvlmaleimide 3.3

Testes

Homogenization conditions BzArgOEt hydrolysis units/g testes

0.5 M KC1 195 0.5 M KCl, 0.25 M benzamidine 188 0.1 M formate, pH 3.0 205

EGTA, ethylene glycol bis(P-aminoethy1 ether)N,N,N'N"tetraa- cetic acid.

4816 Sea Urchin Acrosin

U I O 50 90 130 170 2io

Fraction Number FIG. 1. Chromatography of dialyzed testes extract (300 ml)

on SP-Sephadex C-25. The column (2.75 X 30 cm) was equilibrated with 0.05 M sodium formate, 0.15 M NaCI, pH 3, and eluted at 25 ml/ h. When all of the sample was loaded, the column was washed with 400 ml of equilibration buffer. A linear gradient of 500 ml of 0.15 M NaCl in 0.05 M sodium formate (pH 3) to 500 ml of 0.75 M NaCl in the same buffer was applied. Fractions 1 to 100 were 7.5 ml each, while 101 to 230 were 5 ml each. Fractions 135 to 166 were pooled for use in further experiments. Inset, SDS-polyacrylamide gel electrophoresis of the pooled enzyme in the presence (right) or absence of 1% p- mercaptoethanol. The positions of standard proteins and the molecular weights of the major protein bands are indicated.

6000 37K 34K Dye J . 6 c .e 5000 1 61.5K II

n E J. II o 40004 I I

I 1 . - 30001

., 4 8 12 16 20 24 28 32 36

Slice Number FIG. 2. Distribution of radioactivity in SDS-polyacrylamide

slab gels (in 1% 8-mercaptoethanol) of ["CIDIP-enzyme from testes (- - -) and ["CIDIP-enzyme treated with dimethyl suberimidate (-). The amount of radioactivity in gel slices (2.4 mm each) was determined as described under "Materials and Methods." Molecular weights were obtained by interpolation of the mobility of standard proteins.

ciated with the 34,000-dalton protein (Fig. 2, broken line). When the active enzyme and ['4C]DIP-enzyme were mixed

and applied to a Sephadex G-100 column at pH 3, the two species co-eluted (Fig. 3). A similar elution profile was obtained when the column was equilibrated at pH 6 in the presence or absence of 1% Triton X-100. Calibration of the column, using proteins of known molecular weight, revealed that the testes enzyme eluted with an apparent molecular weight of 53,000. Since the sum of the molecular weights of the two major proteins seen on SDS-polyacrylamide gels is 52,000, it appears that the enzyme may exist as a complex of two subunits, the larger of which (34,000 daltons) reacts with DFP and appears to be the catalytic entity of the enzyme.

Further evidence for the oligomeric nature of this protein was obtained by experiments using dimethyl suberimidate, a bifunctional reagent which has been shown to cross-link pro- tomers in dilute solutions of oligomeric proteins (24). Treat- ment of the [I4C]DIP enzyme with dimethyl suberimidate yielded, by SDS-polyacrylamide gel analysis, a labeled band of M , = 61,500 containing 55% of the radioactivity (Fig. 2). The remaining label migrated with M, = 3,000 larger than the

untreated protein, probably due to intrasubunit reactions of dimethyl suberimidate (MI = 273). Intrasubunit side reactions were also observed with a hemoglobin control treated with dimethyl suberimidate, which yielded not only various cross- linked species of hemoglobin, but also monomer molecular weights ranging from 16,000 to 17,500.

If the testes enzyme is composed of two subunits of 34,000 and 18,000 daltons, then a cross-linked species of M, = 52,000 should be observed. The protein of 61,500 daltons is probably this species. The additional molecular weight may be ac- counted for by attached reagent or by anomalous behavior of the cross-linked protein on SDS-polyacrylamide gels.

The partially purified enzyme was found to be inhibited by small protease inhibitors such as DFP (13 mM), but not by large ones such as ST1 (0.5 mg/ml). Since the enzymatic activity which is exposed in sperm by treatment with egg jelly coat is inhibited by ST1 (16) and this partially purified enzyme is not, it was necessary to compare the enzyme from testes with the one in sperm.

Purification of Enzyme from Sperm Sperm were disrupted in a VirTis homogenizer and sub-

jected to the same procedures as described above for the enzyme derived from testes. Of the enzyme activity observed in the crude homogenate of sperm, 70% was recovered from the SP-Sephadex column as compared to 55% for the testes homogenate. In their general properties, the two enzymes obtained by these techniques appear to be identical. They eluted at similar positions from the Sephadex G-100 column at pH 3, indicating a molecular weight of about 53,000. Both enzymes were inhibited by DFP. Since neither the sperm enzyme nor the testes enzyme was inhibited by STI, we re- examined our purification procedure to seek an enzyme with the same sensitivity to inhibition as that seen in the intact sperm (16).

Purification and Properties of a 34,000-dalton Protease Zso- luted from Sperm

Sperm (100 ml) were homogenized in 3 volumes of cold 0.5 M KCl, 0.2 M sodium formate, pH 3, using a Dounce apparatus. A dialyzed extract (see "Materials and Methods") was applied to an SP-Sephadex (3-25 column at pH 3 (Fig. 4A). As with the earlier preparations, this enzyme exhibited a molecular weight of 53,000 on Sephadex G-100 at pH 3 and was not inhibited by STI.

The pooled fractions from the ion exchange column were

r

- 0 e,-, 12 20 28 36 44 52 60 68

Fraction Number FIG. 3. Chromatography of a mixture of active enzyme and

["C]DIF"enzyme from testes on a column (1.5 X 87 cm) of Sephadex G-100 in 0.05 M sodium formate, 1 M NaCl, pH 3. Fractions of 3 ml were collected at 10 ml/h and analyzed both for hydrolytic activity toward BzArgOEt and for radioactivity.

Sea Urchin Acrosin 4817

adjusted to pH 7 with 2 M Tris and precipitated by the addition of 4 volumes of saturated ammonium sulfate. The mixture was kept on ice for 30 min and centrifuged, and the pellet was resuspended in 5 ml of 10 m~ glycine, 1 M NaCl at pH 2.5. (Alternatively, the enzyme can be concentrated using an Amicon apparatus with a UM-10 membrane.) The resuspended pellet was centrifuged and the supernatant was applied to a Sephadex G-100 column equilibrated at pH 2.5 (Fig. 4B). Most of the enzyme eluted at a position corresponding to a molecular weight of 34,000, but some activity was associated with higher molecular weight material. Apparently, gel fiitra- tion at pH 2.5 (rather than 3.0) separates the 34,000-dalton catalytic unit from the 18,000-dalton protein. The latter was not recovered.

The proportion of the activity eluting at the 34,000-dalton region varied for unknown reasons from one preparation to

II

5 65 75 85 95 105

12 20 28 36 44 52 60

.- cn cn x 0 73

I x

w 0

- 1

c

2 6 10 14 18 22 26 Fraction Number

FIG. 4. Purification of the 34,000 enzyme species. A, chromatography of dialyzed sperm extract (146 units in 320 ml) on SP- Sephadex C-25. The column (1.5 X 6 cm) was equilibrated with 10 mM sodium formate, 0.1 M NaCl, pH 3, at 15 to 20 ml/h, and 4.5-ml fractions were collected. At the arrow, the column was eluted with 10 mM sodium formate, 0.7 M NaCI, pH 3. The bar indicates fractions pooled for further purification. B, chromatography of the resuspended ammonium sulfate precipitate on Sephadex GI00 equilibrated with 10 mM glycine, 1 M NaCl, pH 2.5. The column (1.5 X 86 cm) was eluted at 15 ml/h and 1.8-ml fractions were collected. Fractions were pooled (bar) for further purification. C, affinity chromatography, on STI-Sepharose, of the fractions pooled from the Sephadex G-100 column. The pooled fractions were adjusted to pH 8 and applied to the column (0.7 X 2 cm) previously equilibrated with 10 mM Tris, 1 M NaC1, pH 8, at 15 d / h . The column was washed with the equilibration buffer, then with 10 m~ Tris, 0.25 M NaC1, 20% glycerol, pH 8. The enzyme was eluted with 10 m~ sodium formate, 0.25 M NaC1, 20% glycerol, pH 3. Fractions 1 to 6 were 3 ml each and 17 to 26 were 1.2 ml each. Fractions 19 to 24 were pooled.

TABLE I1 Summary ofpurification of acrosin-like enzyme - . _ .

mg -fold units/mg % units Homogenate of sperm

4.8 7.2 52 78 11 SP-Sephadex chromatog- 1.1 1.6 97 146 90 Dialyzed supernatant 1 1.5 100 151 102

Ammonium sulfate ppt. 5.6 38 25

270 >405 6 8.1 <.02 STI-Sepharose chroma-

2.1 3.2 9 14 4.3 Sephadex G-100 chroma- 4.6 6.9

raPhY

tography

tography

another. Typically, 50 to 60% of the activity was found to have this mobility, but occasionally as little as 20%. When the active fraction of higher molecular weight was concentrated and passed through the same column, the activity was redis- tributed between two peaks as shown in Fig. 4B. When the M, = 34,000 species was chromatographed on a standardized Sephadex G-100 column at pH 3, it eluted with an apparent molecular weight of 34,000 and was inhibited by STI. Thus, it became possible to purify the enzyme further by affinity chromatography using ST1 as a ligand.

Affinity Chromatography The active fractions (34,000 daltons) from the Sephadex

column were adjusted to pH 8 with Tris and applied to a column of ST1 linked to e-aminohexanoyl-Sepharose (Fig. 4C). After washing unbound material off the column, the enzyme was eluted at pH 3. The inclusion of 20% glycerol stabilized the enzyme somewhat but systematic trials to sta- bilize the enzyme for long term storage have not been carried out. The purified enzyme has a half-life of 1% days when stored at 4°C in the eluting buffer. Because of this instability, the partially purified enzyme eluted from the Sephadex column (Fig. 4B) was more suitable for examination of the characteristics of the 34,000-dalton species.

Table I1 summarizes a purification from 100 ml of "dry" sperm (from 15 sea urchins). In the best preparations, it was possible to obtain approximately 50 pg of enzyme with an overall yield of 20%.

Properties of the M, = 34,000 and 53,000 Enzyme Species from Sperm

Rate measurements of the hydrolysis of BzArgOEt by the M, = 53,000 species yielded a K , of 0.6 mM; the corresponding value for hydrolysis by the 34,000 species is 0.2 m ~ , suggesting that the removal of the 18,000-dalton subunit does not alter greatly the binding of this ester substrate. The optimum pH for ester hydrolysis is 7.0 to 7.2 for both enzyme species.

The specific activity of the M, = 53,000 enzyme was determined to be 5.6 units/nmol, using the incorporation of ["]- DFP to define the concentration of active enzyme.

To determine whether either form of the enzyme was a glycoprotein, its affinity for concanavalin A-Sepharose was assessed. The M, = 53,000 enzyme was bound and it eluted with 0.2 M a-methylmannoside. When the column was first incubated with the sugar, less than 5% of the applied enzyme was bound. These results indicate that the interaction between the enzyme and the lectin is specific and that the enzyme contains glucose or mannose units (25). In contrast, the isolated M, = 34,000 enzyme does not bind to concanavalin A- Sepharose, suggesting that only the M, = 18,000 subunit contains the carbohydrate residues necessary for interactions with concanavalin A.


Inhibitors of Esterase Activity Tables I11 and IV show that the BzArgOEt esterase activi-

ties of the M , = 53,000 and 34,000 enzyme species are similarly inhibited by relatively small compounds such as DFP, PMSF, p-nitrophenyl-p'-guanidinobenzoate, or p-aminobenzamidine. However, important differences are observed when larger inhibitors are tested. The M, = 34,000 species is inhibited by proteinaceous inhibitors such as STI, pancreatic trypsin inhibitor, or ovomucoid, as well as by the peptide leupeptin (Table IV), but the M, = 53,000 species is not (Table 111). Although the M , = 53,000 species (which includes the 18,000-

TABLE I11 Inhibitors of esterase activity of the M, = 53,000 enzyme

Enzyme (0.3 to 0.4 unit, partially purified from sperm) was incubated at room temperature (pH 8) in the presence of the stated concentration of inhibitor for 20 min. [3H]BzArgOEt (1.25 m) was then added and the assay was performed as described in the text.

Inhibitor Concentration Per cent inhibition

None PMSF PMSF DFP DFP NPGB" NPGB Benzamidine Benzamidine p-Aminobenzamidine p-Aminobenzamidine TLCK TPCK ST1 Ovomucoid Pancreatic trypsin inhibitor Leupeptin

3.3 mM 10 lllM 2.7 r n ~ 13 m

0.67 m~ 13 m~ 133 m

133 mM

1 mM 0.5 mg/ml 1.7 mg/ml 1.7 mg/ml

0.13 mM

6.7 mM

1 mM

300 M/ml

0 27 70 90 100 40 79 42 89 42 90 0 0 0 0 0 0

" NPGB = p-nitrophenyl-p'-guanidinobenzoate; TLCK = a-L-toluenesulfonyl lysine chloromethyl ketone; TPCK = a-L-toluenesulfonyl phenylalanine chloromethyl ketone.

TABLE IV Inhibitors of esterase andprotease activity of the M, = 34,000

enzyme Details and abbreviations are given in Table 111.

Per cent inhibition of

Esterase Protease activity activity

Inhibitor Concentration

None PMSF DFP DFP DFP NPGB NPGB NPGB TLCK TLCK TPCK p-Aminobenzami-

ST1 dine

Ovomucoid Pancreatic trypsin

inhibitor Leupeptin Leupeptin Leupeptin Chymostatin Chymostatin

0 100 42 76

46

76

100

76

100 99 86

100

0

100

50

32

10 93

100 100 100

10 49

62 81

TABLE V Digestion of ['4C]lysozyme by sperm enzymes

Enzyme Relative ester- Trichloroacetic ase activity acid-soluble

Source M , counts/5 min

Sperm 34 ,000 1.1 1099 Sperm 53,000 1.0 90 Testes 53,000 1.2 85 No enzyme 0 104

TABLE VI Peptides isolated after digestion of bovine insulin B chain

Numbers refer to ratios of amino acids; numbers in parentheses refer to those in bovine insulin B chain corresponding to the residues at the bottom

Peptide

A C D E

Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Mobility" Yield Residue numbersb

1.03 (1)

0.94 (1)

0.94 (1)

0.98 (1)

1.05 (1)

+0.43 65% 26-30

0.7 (1) 0.7 (1)

0.4 (1) 2.3 (2)

1.2 (1) 1.0 (1) 1.9 (2) 3.1 (3) 0.5 (1) 1.4 (1) 1.4 (2)

-0.16 21%

1-16

0.8 (1)

1.3 (1)

1.7 (2)

1.1 (1) 1.2 (1)

2.0 ( 2 )

1.0 (1) -0.22 33% 17-25

0.7 (1)

1.6 (1)

1.1 (1)

0.9 (1) 1.1 (1)

1.0 (1) -0.29 18% 17-22

" Mobility during electrophoresis at pH 6.5 where markers of cysteic acid, alanine, and lysine have mobilities of -1.0, 0, and +1.0, respec- tively.

See Ref. 26.

dalton protein) has esterase activity similar to that of the M , = 34,000 species, it appears to lack the ability to interact with protein protease inhibitors, as well as protein substrates (see below).

Digestion of Proteins by Purified Enzymes Digestion of ['4C]Carboxamidornethyl Lysozyme-The

proteolytic activity of the enzyme preparations were examined using ['4C]lysozyme as the substrate. Only about 30% of the radioactivity was solubilized in 18 h, either because the substrate (or a digestion product) is quite insoluble at neutral pH, or because the protease activity was gradually lost during the course of the reaction. Significantly, digestion was completely inhibited by ST1 (1 mg/ml) as well as by a variety of other inhibitors of serine proteases (Table IV).

In contrast, when ['4C]lysozyme was treated with the M , = 53,000 species from either testes or sperm, no radioactivity was solubilized (Table V). Therefore, it appears that the 18,000-dalton protein inhibits protease activity but not esterase activity.

Digestion of Oxidized B Chain of Insulin-Digestion of 50 nmol with 0.23 unit of the M, = 34,000 enzyme yielded a pattern of peptides indicating that the M, = 34,000 protease cleaves several bonds in insulin. The peptides were eluted with 0.1 M NH40H, and their amino acid compositions were determined. In Table VI, these results are identified with known sequences of insulin.

Peptide A is in highest yield, indicating that the Phe-Tym linkage is hydrolyzed with the highest efficiency. Peptide C

Sea Urchin Acrosin

represents the NHz-terminal region cleaved at Tyr-Leti,-/ and isolated in about one-third the yield of Peptide A. Peptides D and E both must have leucine-17 at their NH2 termini. Peptide D continues to phenylalanine-25, whereas Peptide E (in 18% yield) resulted from cleavage of the Arg-Glyts bond.

The protease cleaved on the carboxyl side of tyrosine-16 and phenylalanine-25 to a greater extent than arginine-22. No cleavage after lysine-29 was observed. Bovine trypsin (26) and boar acrosin (5,11) cleave insulin after arginine-22 and lysine- 29 whereas chymotrypsin cleaves tyrosines-16 and -26 and phenylalanine-25 (26). Therefore, it appears that the preparation of sea urchin sperm protease possesses both trypsin- and chymotrypsin-like activities. Alternatively, two enzymes could be present, one possessing trypsin-like activity, the other resembling chymotrypsin in specificity. However, the proteolytic activity of the M, = 34,000 enzyme toward [‘%]lysozyme can be inhibited by p-aminobenzamidine, p-nitrophenyl-p‘- guanidinobenzoate, and leupeptin (all inhibitors of trypsin- like enzymes) as well as by chymostatin, an inhibitor of chymotrypsin (Table IV). This suggests that one enzyme possessing a rather broad specificity is responsible for the observed digestion.

Isolation from Supernatants ofdcrosome-reacted Sperm- When sea urchin sperm undergo the acrosome reaction, approximately 20% of the total esterase activity is released from the sperm into the surrounding seawater (17). Since this released enzyme is inhibited by STI, the enzyme could be purified in one step by application of the supernatant to an affinity column of STI-Sepharose.

A concentrated supernatant from 10 ml of acrosome-reacted sperm was applied to an STI-Sepharose column (as in Fig. 4C), the column was washed, and the enzyme was eluted with 50 m&r sodium fox-mate, 0.5 M NaCl, pH 3. Approximately two- thirds of the enzyme activity bound to the column and was eluted at pH 3. This purified enzyme is inhibited by 6 DIM

DFP and by 0.7 mg/ml of STI. Because of the relatively small amount of enzyme released from the sperm, and the large dilutions of sperm necessary to trigger the acrosome reaction, this method was not practical for the purification of the enzyme. No further characterization of this enzyme was carried out.

DISCUSSION

This report describes an enzyme, isolated from homogenates of sea urchin sperm or testes, which catalyzes the hydrolysis of benzoylarginine ethyl ester and is inhibited by DFP. In these respects, it resembles many serine proteases including acrosin from vertebrate sperm, trypsin from pan- creas, and coagulation factors from blood plasma. However, this 53,000-dalton enzyme has several unusual characteristics which, on the one hand, suggest a role in the fertilization process, and, on the other hand, imply a pattern of regulation in expression of its enzymatic function.

It has already been reported that the hydrolytic activity toward BzArgOEt is shielded in intact sea urchin sperm and “exposed” to substrate by treatment of the sperm with dilute extracts of egg jelly coat (16). Since homogenates of sperm display this activity, intact sperm may simply be refractory to substrate entry. The homogenates contain a 53,000-dalton enzyme which can be dissociated, at pH 2.5, into a 34,000- dalton catalytic subunit and an 18,000-dalton subunit. Al- though both the M, = 53,000 and 34,000 forms of the enzyme catalyze the hydrolysis of BzArgOEt and are inhibited by DFP, only the M, = 34,000 form displays proteolytic activity and is susceptible to large proteinaceous inhibitors.

In several respects, the protease in sea urchin sperm bears an analogous relationship to acrosin in vertebrate sperm (2,

53 K enzyme Actwe vs. BzArg OEt only

n 34 Ki

pti 2 5

u I

n Y _--_

I

0 x 1

34 K subunIt Active Protease Actwe YS EizArg OEt mhlblted by STI

J

FIG. 5. A diagrammatic model illustrating characteristics of two forms of the acrosin-like enzyme. On the Zeft is the M, = 53,000 (53 K) form of the enzyme with the M, = 18,000 (18 K) subunit (the shaded “doughnut”) masking much of the surface of the M, = 34,000 (34 ZC) subunit (the large square). A portion of the active site (X) is accessible to small substrates (BzArgOEt) and inhibitors (DFP or PMSF). Dissociation at pH 2.5 separates the two subunits (on the right). In the dissociated it4, = 34,000 subunit, it is postulated that the exposure of the periphery (Y and 2) of the active site (X) permits the approach of protein substrates or inhibitory proteins. The geometry of the sketch is entirely arbitrary. Other models of partial occlusion would also tit the observations.

10, 11). Both are serine proteases, inhibited by DFP and STI. Both activities appear during the fertilization event. Acrosin has been localized in the acrosomal region of mammalian sperm (2). Although we do not yet have corresponding data for the sea urchin protease, it is exposed by treatments which induce formation of the acrosomal filament (16, 17). In both cases, inhibition of the protease prevents induction of the acrosomal filament (17, 27). However, vertebrate acrosins have been found as zymogen precursors (12-15), whereas no indication of a zymogen form is found in sea urchin sperm. But in a sense the 53,000 form of the enzyme is the functional equivalent of a zymogen: it lacks protease activity in its oligomeric form and removal of the 18,000 subunit generates protease activity.

Thus, it becomes important to identify whether the enzyme is in the M, = 34,000 or the 53,000 form as sperm encounter eggs. These studies have not yet been performed, but prelim- inary experiments indicate that the enzyme functions as the M, = 34,000 species in homogenates of sperm which have been pre-exposed to egg jelly coat.’ Together with the finding that the activity toward BzArgOEt is crucial to the morphological change known as the “acrosome reaction,” these observations imply that the enzymatic activity may participate in a cascade of regulatory events during the early stages of the fertilization process.

The nature of the M, = 53,000 form of the enzyme presents a paradox. It is a fully active esterase, susceptible to inhibition by compounds of low but not high molecular weight, and it lacks protease activity toward insulin and lysozyme. The removal of the M, = 18,000 subunit has little effect on the esterase activity of the M, = 34,000 subunit or its susceptibility to small inhibitors, but a profound effect on its interactions with large substrates or inhibitors. Precedent for such selec- tivity is found in the interaction of proteases with cy2-macroglobulin, wherein the protease retains its activity toward small substrates but is inhibited in further interactions with large ones (28). However, cy2-macroglobulin is very large (-725,000 daltons) and is thought to envelop a protease in a network which filters out large substrate molecules. The 18,000-dalton subunit is much smaller and it is unlikely that a similar explanation can account for the nature of the M, = 53,000 protease. Instead, the model in Fig. 5 is proposed, where the M, = 18,000 subunit occludes secondary binding sites and

’ A. E. Levine and K. A. Walsh, unpublished data.


prevents the approach of large molecules to the M, = 34,000 subunit. While we have no evidence to support the specific geometry of this model, it fits conceptually the experimental data.

Alternatively, the M, = 53,000 species could be an enzyme. inhibitor complex. Acrosin inhibitors are present in vertebrate seminal fluid (29) and possibly also associated with the sperm. An acrosin-inhibitor complex (which can be dissociated at pH 3) is present in homogenates of mammalian sperm. Subcellular localization of the M, = 34,000 and 18,000 species in sea urchin sperm is needed to indicate whether the two proteins could be associated in vivo. If the two proteins are not in the same subcellular compartment their association would be an arti- fact of the homogenization. However, if the M , = 53,000 species is an enzyme. inhibitor complex which dissociates during the acrosome reaction, it presents an interesting model of an activation mechanism.

Early studies of the esterase activity toward BzArgOEt in sea urchin sperm homogenates indicated a crucial difference from the corresponding activity in activated sperm. Whereas both activities are inhibited by DFP, the activity in the sperm but not the homogenate was inhibited by STI. These observations suggested that the enzyme as isolated from the homogenate was not the active principle in the cell. The two- subunit model offers an explanation of these findings and raises the unanswered question of the intracellular relationship of the M , = 18,000 and 34,000 subunits during sperm activation.

The specificity of the 34,000 enzyme appears to be somewhat unusual. The enzyme hydrolyzes peptide bonds in the B chain of insulin which correspond to the substrate specificity of both chymotrypsin and trypsin. The enzyme is inhibited by various trypsin inhibitors (Table IV) and by chymostatin (an inhibitor of chymotrypsin). Possibly, secondary binding sites are important in defining the specificity of this enzyme. An enzyme from crab hepatopancreas has similar broad specificity (30) and bovine trypsin itself possesses minor chymotrypsin-like activity (31).

The role of the sperm protease in the fertilization process is unclear. It has been proposed that acrosin is involved in penetration of outer coats surrounding the egg (1, 2). If one assumes that all of the protease is in the acrosomal granule of sea urchin sperm, its concentration would be 0.7 mM or 240 mg/ml," a potentially powerful lytic device. Similar concentrations have been calculated in mammalian sperm (29). It should be noted that small holes are observed in egg coats after sperm penetration (29, 32), whereas soluble acrosin to- tally digests mammalian zona pellucida (9, 29). Alternatively, the protease could serve more subtle control functions by limited proteolysis of precursor proteins before or after sperm/ egg fusion. Possibly, its role in induction of the acrosome reaction is of this type (17). Further study of the localization, control, and specificity of this protease should clarify new molecular interactions in the fertilization process.

'Calculated assuming that each sperm has 3 X 10"' units of enzyme of specific activity 5 units/nmol and the acrosomal granule is a sphere of diameter 0.25 p (1) .

Acknowledgments-We wish to thank Drs. Hans Neurath and John Lo,mdale-Eccles for their helpful suggestions and Eric Fodor for his efforts in the initial phases of this work.

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