DEVELOPMENTAL
BIOLOGY
Isolation
44, 22-32 (1975)
and Biological Sea Urchin EDWARD
Scripps Institution
of Oceanography,
Activity
of the Proteases
Eggs following
J. CARROLL,
JR.
Fertilization
AND DAVID
Marine Biology Research Division, La Jolla, California 92037 Accepted
Released by
EPEL Uniuersity
of California,
San Diego,
December 23, 1974
The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity on p-aminobenzamidine-Sepharose columns. Elution profiles from affinity chromatography columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low PH. The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between lo5 and lOa were demonstrated by gel filtration. INTRODUCTION
Sea urchin eggs release macromolecules into the surrounding seawater following fertilization. This solution of macromolecules is referred to as the fertilization product (Ishihara, 1964) and is primarily composed of the soluble cortical granule exudate. The exudate contains protease activity which is sensitive to soybean trypsin inhibitor’ (Vacquier et al., 1972). The function of this activity has been investigated by fertilizing eggs in the presence of STI. Two processes are prevented: (1) The elevation of the fertilization membrane (Hagstrom, 1956; Liinning, 1967; Vacquier et al., 1972; Schuel et al., 1973) and (2) the detachment of excess sperm ‘The abbreviation inhibitor.
used is: STI, soybean trypsin
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
(Vacquier et al., 1973a). Such eggs also become polyspermic (Vacquier et al., 1972; Schuel et al., 1973), suggesting that the protease activity is also involved in the block against polyspermy. The role of this enzyme activity has also been investigated by incubating eggs in the fertilization product. This treatment modifies the egg surface and also prevents subsequent fertilization (Vacquier et al., 1973a). This loss of fertilizability (fertilization-inhibitor activity) is associated with an inability of sperm to attach to the vitelline layer, most likely from alteration of proteinaceous sperm receptors on this layer (Aketa, 1967). Because both fertilization-inhibitor activity and protease activity are prevented by ST1 and because both have similar heat inactivation curves, it was postulated that both activities were properties of the same pro-
CARROLL AND EPEL
tein (Vacquier et al., 1973b). In this paper we report our progress on the purification of the protease activity of the fertilization product. Polyacylamidegel electrofocusing revealed two proteases in the fertilization product. These proteases, partially resolved into two fractions by affinity chromatography, had dramatically different biological activities. One fraction alters the vitelline layer such that sperm cannot bind to and fertilize eggs; we shall refer to this activity as “sperm receptor hydrolase”. The other protease fraction’ is involved in vitelline layer elevation through cleaving connections between the vitelline layer and egg surface; we shall refer to this activity as “vitelline delaminase activity”. MATERIALS
AND
METHODS
Preparation of gametes. Shedding of gametes was induced in the sea urchin Strongylocentrotus purpuratus by injection of 0.5 A4 KC1 into the coelomoic cavity. Sperm were collected “dry” and stored at 0°C. Egg jelly was removed by washing eggs for 3 min in seawater which was acidified to pH 5.0 with 1 N HCl or by sieving egg suspensions five times through No. 183 Nitex mesh. The extent of dejellying was ascertained by gentle hand centrifugation of a portion of the egg suspension. The presence of a homogenous pellet was taken as the endpoint. Quantitation of egg and sperm concentrations was done according to Vacquier and Payne (1974). Bioaways. Sperm receptor hydrolase activity was assayed using inhibition of fertilization as the criterion, as described by Vacquier et al. (1973a) with minor modifications. The egg concentration used was 500/ml. Just before use, sperm suspensions were diluted in seawater from “dry” semen to an absorbance at 340 nm of 1.0 (approximately 1.20 x lo8 sperm/ml). To 200 ~1 of egg suspension was added 50 ~1 of the fraction to be tested; the suspension was incubated for 30 min at 16°C. Following
Sea Urchin
Egg Protease
23
this incubation, 50 ~1 of sperm suspension was added and the extent of fertiliz; ation determined by scoring for the presence of hyaline layers 30 min postinsemination and for cleavage at 90 min post-insemination. The assay of vitelline delaminase activity was as described by Carroll and Epel (1974). The presence of activity is indicated by normal elevation of a fertilization membrane when eggs are activated in the presence of STI. A small number (ca 100) of dejellied S. purpuratus eggs are incubated with the fraction to be tested in a covered Syracuse dish at 16°C. At the conclusion of the incubation, ST1 and the parthenogenetic agent, ionophore A23187 (Steinhardt and Epel, 1974), were added to final concentrations of 0.37 mg/ml and 5 PM, respectively. Controls of no fraction and solvent (dimethyl sulfoxide) were run concurrently. The eggs were then immediately scored for the presence of elevated vitelline layers. Preparation of fertilization product. The method of Vacquier et al. (1973a) was used, with minor modification. Fertilization was accomplished by mixing a 50% (V/v) egg suspension with an equal volume of sperm suspension containing 3 ml of “dry” semen for each 25 ml of packed egg cell volume. All manipulations of fertilization product preparations and purified fractions were done in 0.01-0.10 M sodium acetate, pH 4.0, to prevent “activation”, as described in Results. Manipulations of preparations requiring exposure to neutral or alkaline pH were accomplished as quickly as possible. Enzyme assays. Esterase activity was measured according to Rick (1963) except that reaction mixtures contained 60 mM Tris-HCl, 0.5 mM a-N-benzoyl-r.-arginine ethyl ester at pH 8.0. Assays were run at ambient temperature. The Ki of the esterase activity for p-aminobenzamidine was determined from Lineweaver-Burk plots of data obtained from assays at ten substrate
24
DEVELOPMENTALBIOLOGY
concentrations in the presence and absence of 1.36 x lo-‘M p-aminobenzamidine. Sucrose gradient centrifugation. This was accomplished as described by Martin and Ames (1961) using 5-20s linear sucrose gradients containing 0.1 M Tris-HCl0.4 M NaCl, pH 8.0. Standards were Escherichia coli alkaline phosphatase and bovine hemoglobin. Sedimentation rates were calculated using the published sedimentation coefficients for alkaline phosphatase (Garen and Levinthal, 1960) and bovine hemoglobin (Edsall, 1953). Alkaline phosphatase activity was assayed by incubating aliquots of fractions in 1.0 ml of 1.0 mM p-nitrophenyl phosphate-O.1 M Tris-HCl at pH 8.0 and 37°C for 45 min. The reaction mixture was diluted to 3 ml with water and read at 412 nm in a Gilford Model 300N rapid sampler calorimeter. Electrofocusing. Analytical electrofocusing was essentially according to Wellner (1971) using final concentrations of 4.8% acrylamide, 0.25% N,N,N’,N’-tetramethylethylenediamine, and l-1.4% pH 3-5 ampholines plus 0.6% pH 3-10 ampholines. The anodic solution contained 0.96% H,PO, and the cathodic solution contained 1.7% ethylenediamine.
VOLUME 44, 1975
justed to 4.9 with NaOH. The coupling reaction was initiated by the addition of 1.7 g of 1-cyclohexyl-3-(2-morpholino-ethyl)carbodiimide metho-p-toluene-sulfonate. The pH was maintained at 4.6-4.9 for 5.5 hr at which time the gel was exhaustively washed with water and resuspended in 0.1 M Tris-HCl-0.4 M NaCl, pH 8.0. Protein concentrations were estimated according to either Lowry et al. (1951) or Warburg and Christian (1941). All reagents were prepared from commercially available products with deionized, glass-distilled water. RESULTS
Purification tilization
and Biological Activity of FerProduct E&erase Actiuity
Isoelectric precipitation. The first step in purification was isolation of the enzyme from seawater. When preparations of fertilization product were adjusted to pH 4.0, a precipitate formed which was enriched in esterase activity; a typical preparation is presented in Table 1. The esterase activity was found only in the pH 4.0-insoluble fraction. In various preparations the specific activity was increased 1.3- to 4-fold with an average recovery of 106% (seven Preparation of p-aminobenzamidinepreparations). Assays for sperm receptor CH-Sepharose 4B. Four grams of CH- hydrolase activity indicated that this acSepharose 4B (Pharmacia) were hydrated tivity was also located in the pH 4.0-insoluand washed exhaustively in 0.5 M NaCl. ble fraction. The pH 4.0-supernatant soluThe gel was adjusted to 10% (V/V) in the tion had no detectable effect on fertilizasame medium and 160 mg of p-aminobenz- tion. amidine .2HCl added and the pH adSince cortical granules also contain pTABLE
1
ISOELECTRICPRECIPITATION OF ESTERASE ACTIVITY IN FERTILIZATION PRODUCT Fraction
Crude fertilization
product*
pH 4.0-insoluble fraction’ pH 4.0~soluble fraction
IU”
Mg
IU/mg
8.4
345
0.0243
-
7.2 0.24
224 121
0.0321 0.002
85.7 2.9
Total recovery (percent)
“The esterase assay as described in Materials and Methods was used. * Fertilization product was prepared as described in Materials and Methods. c Precipitation at pH 4.0 was accomplished as described in Results.
Percent of activity in fraction
Purification (n-fold) -
97 3
1.3 -
CARROLLANDEPEL
glucanase activity (Schuel et al ., 1972)) the fractions obtained by isoelectric precipitation were examined for the presence of this activity. The pH 4.0-supernatant fraction contained 96% of the /3-glucanase activity recovered, the remaining 4% was contained in the pH 4.0-precipitate fraction. Affinity chromatography. Initial attempts at affinity chromatography made use of Sepharose resin prepared according to Robinson et al. (1971) with ST1 substituted for ovomucoid. Protease activity of the unfractionated fertilization product or the pH 4.0-insoluble fraction was firmly retained on a column of this resin. However, elution with a pH change (pH 10.5) or ru-N-benzoyl-L-arginine ethyl ester containing solutions (0.05-0.10 M at pH 8.0) was not successful; thus STI-Sepharose was unsuitable as an affinity adsorbent. The competitive trypsin inhibitor paminobenzamidine (Mares-Guia and Shaw, 1965) was tested as a possible ligand for an affinity resin. With the pH 4.0insoluble fraction (dissolved in 0.1 M Tris-HCl, pH 8.0) the K, of p-aminobenzamidine, using a-N-benzoyl-L-arginine ethyl ester as substrate, was 1.6 x 1O-4M. Thus p-aminobenzamidine was a sufficiently good inhibitor to make affinity chromatographic methods feasible. The various batches of p-aminobenzamidineCH-Sepharose 4B resin that were prepared did not adsorb detectable quantities of either commercial chymotrypsin or trypsinogen (based on absorbance at 280 nm). Commercial trypsin was tightly bound to the extent of 3-7 mg of trypsin/ml of resin (based on absorbance at 280 nm) and could be specifically eluted with cY-N-benzoyl-Larginine ethyl ester or p-aminobenzamidine-containing solutions (0.1-20 mM, pH 8.0). Elution of trypsin was also accomplished using either acidic or basic pH changes (pH 3.0 or 11.6). The esterase activity of the pH 4.0insoluble fraction was also bound by this matrix. Attempts at elution of bound pro-
Sea Urchin Egg Protease
25
tein using p-aminobenzamidine (1 .Oto 100 mM, pH 8.0) or cu-N-benzoyl-L-arginine ethyl ester-containing solutions (1.0-250 mM, pH 8.0) met with limited success, viz, the eluted protein was dilute and recovered in poor yield. Elution of bound enzyme was best accomplished using a basic pH change. A preparation of fertilization product purified by isoelectric precipitation and containing 3.7 international units (IU) of esterase activity and 24 mg of protein (in 0.1 M Tris-HCl-0.4 M NaCl, pH 8.0) was applied to a p-aminobenzamidineSepharose column at a flow rate of 450 ml/hr. Under these conditions, 35% of the applied esterase activity was bound to the column. An elution diagram of the esterase activity and protein eluted with 0.1 M NH,OH-0.4 M NaCl (pH approximately 11.6) is shown in Fig. 1. The esterase activity and protein peaks are incongruent, meaning that the specific activity is not constant across the profile and suggesting the existence or presence of more than one enzyme. The leading and trailing edges of the peak had limiting specific activity values of 0.01 and 0.10 IU/mg, respectively. The degree of separation of protein and esterase activity could be considerably improved by decreasing the flow rate, as shown in Fig. 2. The conditions and enzyme preparation used were virtually identical with those of Fig. 1 except that the flow rate was 60 ml/hr. Using this flow rate, 74% of the esterase activity applied was bound to the column, and in other experiments close to 100%of the esterase activity was bound. Also under these latter conditions almost all of the protein of the pH 4.0-precipitate fraction was bound to the column, indicating that this fraction is composed almost entirely of protease activity. The effect of column dimension was not thoroughly investigated, but the greatest degree of separation was observed when columns had length/diameter ratios of approximately 20.
26
DEVELOPMENTALBIOLOGY
VOLUME 44, 1975
7 6
-10 7 0* -4z 3
-6,”
-2 -
Fraction Number FIG. 1. Chromatography of the pH 4.0-insoluble fraction of fertilization product bound to a p-aminobenzamidine-CH-Sepharose 4B column. A 2.2 x 4-cm column was poured and equilibrated with 0.1 M Tris-HCI-0.4 M NaCl, pH 8.0. A solution containing 3.7 IU of esterase activity and 24 mg of protein was applied to the column in the same buffer. The column was washed with about 15 bed-volumes of the same buffer until the absorbance of the eluate was 10.02. The bound protein was eluted at a flow rate of 450 ml/hr with a 0.1 M NH,OH-0.4 M NaCl solution; the eluate was collected in tubes containing sufficient 1.0 M sodium acetate to maintain the pH at 4.0.0, mg/ml protein; 0, IU/ml esterase activity; x, IU/mg.
0
2
L
, ,
iIO
20
-*---y--.
30 Fraction Number
40
50
FIG. 2. Chromatography of the pH 4.0-insoluble fraction of fertilization product bound to a p-aminobenzamidine-CH-Sepharose 4B column. A 2.2 x 4-cm column was poured, equilibrated, and developed as described in the legend to Fig. 1, except that the flow rate was 60 ml/hr. A solution containing 5.5 IU of esterase activity and 36 mg of protein was applied to the column. 0, mg/ml protein; 0, III/ml esterase activity; x, W/ml.
CARROLL AND EPEL
Polyacrylamide-gel electrofocusing provided direct evidence for the presence of multiple proteins in the preparations purified by isoelectric precipitation and affinity chromatography. Using the combinedactivity fraction from the affinity column (indicated by the bar in Fig. 2), two proteins were resolved with isoelectric points of 4.7 and 4.9 (Fig. 3). Extraction and enzymatic assay of the separated proteins from the gel was not possible since the proteins precipitate at equilibrium and gel extracts contained an insufficient amount of protein for assay. Use of higher levels of input protein diminished resolution, but all enzymatic activity extracted was coincident with protein (Fig. 4). As noted, two functions attributed to the fertilization product protease activity relate to detachment of the vitelline layer and destruction of sperm receptors. As shown, there are at least two. species of protease in the fertilization product. Although complete separation of the proteases was not accomplished on a preparative basis, we made use of the widely differing specific activities across the affinity chromatogram peak (Figs. 1 and 2). Collection of the leading and trailing edges yielded partial separation into a lowspecific-activity fraction and a high-specific-activity fraction. Equivalent amounts of activity (a-N-benzoyl-L-arginine ethyl ester as substrate) were then applied to eggs in bioassay procedures. The results of a typical bioassay are presented in Fig. 5. In the vitelline detaching assay, a positive result is indicated by the presence of normally elevated fertilization membranes upon parthenogenetic activation of the test eggs with ionophore A23187 in the presence of STI. In the absence of this activity (Fig. 5A) the fertilization membrane remains firmly attached to the egg surface at many sites and has a rosette appearance. A positive result in the sperm receptor hydrolase assay is indicated
27
Sea Urchin Egg Protease
I
I
1
I
1
I
0.8 1.0 0.2 0.4 0.6 Fraction of Gel Length FIG. 3. Polyacrylamide-gel electrofocusing of the pH 4.0.insoluble fraction of fertilization product purified by affinity chromatography. Electrofocusing was at 55 V for 17 hr at 4°C; 94 pg of protein were applied. The preparation used was the pooled fractions indicated by the bar in Fig. 2. A parallel gel was frozen on a paraffin block and sliced into 58 fractions; 1 ml of water was added to each slice and the resultant pH measured after 24 hr of extraction.
I
+
FIG. 4. Polyacrylamide-gel electrofocusing and enzymatic activity assay of the pH 4.0~insoluble fraction of fertilization product purified by affinity chromatography. Conditions for electrofocusing and pH determination were as described in the legend to Fig. 3 and in Materials and Methods. Three milligrams of the preparation described in the legend to Fig. 3 were applied to the gel. The schematic representation of the gel was obtained from measurements of the precipitated protein visible in the gel at the end of the run. The gel was then sliced into eight fractions and homogenized in a Dounce apparatus with 0.5 ml of 1.0 M Tris-HCl, pH 8.0, at O”C, per mm of gel slice. Extraction was for 90 min; then the homogenates were centrifuged at 10,000 g for 1 min and the extracts were assayed for esterase activity.
FIG. 5. Biological assays for vitelline detaching activity and sperm receptor hydrolase activity in the lowand high-specific-activity fractions from an-aminobenzamidine-Sepharose-CH 4B column. In a total volume of 250 ~1, 100 eggs were incubated for 30 min at 16°C in each of the various fractions. Assays using the low-specific-activity fraction contained 44 Fg of enzyme at 0.028 IU/mg; assays containing the high-specificactivity fraction contained 12 fig of enzyme at 0.093 IU/mg. Assays using the mixture of fractions contained the same amounts of the enzymes as above. Vitelline detaching assay: Eggs treated with seawater (A), low-specific-activity fraction (B), high-specific-activity fraction (C), a’ mixture of low- and high-specific-activity fractions activated with ionophore A23187 in the presence of ST1 (D). Sperm receptor hydrolase assay: Eggs treated with seawater (E), low-specific-activity fraction (F), high-specific activity fraction (G), a mixture of low- and high-specific-activity fractions and fertilized (H).
by the failure of sperm to bind and fertilize test eggs; in the absence of this activity eggs fertilized normally (Fig. 5E). The low-specific-activity fraction had vitelline detaching activity (Fig. 5B) but no sperm receptor hydrolase activity (Fig. 5F). Conversely, the high-specific-activity fraction had no vitelline detaching activity (Fig. 5C) but had sperm receptor hydrolase activity (Fig. 5G). These two activities are independent, as a mixture of the two fractions exhibited both activities (Figs. 5D and H). These data suggest that these biological activities reside in at least two separate protease molecules. Additional Properties of Fertilization uct E&erase Activity
Prod-
Activation of fertilization product esterase activity. Esterase activity of the fertilization product increases five- to tenfold during storage at 0°C (Vacquier, unpublished observation). This activation becomes apparent by 24 hr and continues for several days. There is concomitent loss of
sperm receptor hydrolase activity as determined by the bioassay. We have also noted that these activated preparations digest the vitelline layer. As sperm receptor hydrolase activity cannot be assayed when the vitelline layer is absent, we do not know if activation directly alters sperm receptor hydrolase activity. Both esterase activity and sperm receptor hydrolase activity remained constant when preparations were stored at O”C, pH 4.5, or at -2O”C, pH 8.4 or pH 4.5, whereas storage at O”C, pH 8.4, resulted in approximately sevenfold activation over a 4-day period (Fig. 6). Accordingly, fertilization product preparations were routinely stored at - 20°C as a suspension in 0.05 M sodium acetate-50% glycerol, pH 4.0-4.5. Preparations were stored in the liquid state (50% glycerol) because rapid dissolution of the precipitate was possible following readjustment of the pH to 8.0. When these precipitates were frozen, dissolution at pH 8.0 proceeded very slowly and activation frequently occurred.
CARROLL AND EPEL
8
16 DAYS
Sea Urchin
24
FIG. 6. Activation of fertilization product esterase activity. Portions of a fertilization product preparation were stored under various conditions and assayed at various times. To one portion was added %Ovolume of 0.5 M sodium acetate, pH 4.0, and to another was pH 8.0. Oneadded SO volume of 0.5 M Tris-HCI, milliter aliquots were stored at 0” or at -22°C and assayed after the indicated time of storage. The final pH’s were 4.45 and 8.35, respectively. q , pH 8.35, -22°C; 0, pH 8.35, 0°C; n , pH 4.45, -22°C; l , pH 4.45. 0°C.
Gel filtration. When the pH 4.0-insoluble fraction was chromatographed on a Sepharose 4B column, multiple peaks of e&erase activity were apparent with approximate molecular weights between lo5 and lo6 (Fig. 7). This heterogeneity of esterase activity is possibly the result of aggregation, since rechromatography of fraction I resulted in the appearance of fractions I and II. The same elution patterns were obtained with several preparations both at pH 5.6 and 8.0. Molecular weight estimation. From velocity sedimentation in sucrose, the single peak of esterase activity was 4.5 S (Fig. 8). This corresponds to a molecular weight of 47,000.
Egg Protease
29
which alters the vitelline layer such that sperm cannot bind to it and cannot fertilize the egg. Therefore, the vitelline layer is a physical barrier to fertilization of the egg, and the fertilizing sperm cell must bind and interact with a specific receptor on the vitelline layer surface prior to penetration of the egg. The high-specific-activity fraction also detaches bound sperm from eggs (Carroll and Epel, unpublished observations) . Destruction of sperm receptors on the vitelline layer and detachment of supernumerary sperm from the vitelline layer are important components in the block against polyspermy (Vacquier et al., 1973a). Aketa (1967) has shown that the vitelline layer contains sperm-binding proteins. Proteolytic action on these molecules by sperm receptor hydrolase before sperm binding would prevent a sperm from binding to the receptor whereas proteolysis of a receptor after binding would result in sperm detachment. Previous work has shown that sperm detachment and fertilization membrane elevation occur simultaneously (Vacquier and Payne, 1974). Since vitelline detaching activity and sperm receptor hydrolase ac-
I 16 P 12
4,;
08
30 : 3 20 -
04
IO
Na
DISCUSSION
In this paper we show that the protease fraction of the fertilization product consists of at least two enzymes. A low-specificactivity fraction contains vitelline detaching activity which apparently removes connections between the plasma membrane and vitelline layer as a part of the elevation process. The high-specific-activity fraction contains sperm receptor hydrolase activity
15
20 25 30 FRACTION NUMBER
35
FIG. 7. Chromatography of the pH 4.0-insoluble fraction of fertilization product on a Sepharose 4B column. A 1.4 x 36-cm column was poured and equilibrated with 0.1 M Tris-citrate at pH 5.6 and 4°C. A solution containing 1.24 IU of esterase activity was applied in the same buffer containing 8% sucrose. The void volume was determined in a separate run with blue dextran as marker. 0, absorbance at 280 nm; 0, IU/ml, esterase activity.
30
DEVELOPMENTAL
VOLUME 44, 1975
BIOLOGY
BOTTOM
TOP
014 -012
006
%
002 ‘0 ‘a
lo-
20 Fracl~on Number
o
30
-
-o--o-o
40
FIG. 8. Sucrose gradient ultracentrifugation of the pH 4.0~insoluble fraction of fertilization product. Two-hundred-forty microliters of a solution containing 0.085 IU of esterase activity (1.3 mg of protein), 2 mg of hemoglobin and 25 rg of E. coli alkaline phosphatase were layered on a 5.4-ml gradient. Centrifugation was for 45 hr at 49,500 rpm in a SW5OL rotor at 4°C. At the end of the run fractions were collected, 10 pl were assayed for alkaline phosphatase activity (0) and 50 ~1 assayed for e&erase activity (O), and 1.5 ml of water was added to the remaining solution and the absorbance at 415 nm was monitored for hemoglobin (A).
tivity apparently reside in different mole‘The proteases of the fertilization product cules, the simultaneous release of two pro- are highly specific enzymes. Bovine trypsin teases from the cortical granules now ex- and various other proteases (Berg, 1967) plains the coincidence of elevation and will digest the vitelline layer such that it sperm detachment. Attachment of the does not elevate at fertilization. The trypplasma membrane to the vitelline layer by sin-like enzymes of the fertilization product, however, do not act in this fashion. protease-sensitive sites is not a prerequisite layer such that for fertilization since delaminated eggs are One alters the vitelline fully fertilizable. These results also suggest sperm do not bind to it, and another that plasma membrane-vitelline layer con- digests points of attachment between the nections are distinct from the sites of plasma membrane and vitelline layer as a part of the elevation process. However, sperm binding and penetration. The complete separation of the two pro- when the esterase activity of the fertilizateases has not yet been realized, and this tion product activates during storage at pH task is a formidable one for several reasons. 8, specificity is lost and its action on the The biological activities are unstable at pH vitelline layer becomes like that of bovine 8.0; activation of esterase activity occurs trypsin in digesting the vitelline layer. The mechanism of this activation is with a concomitant loss in specificity. The presently unclear. Possibly the enzymes enzymes have similar molecular weights and have a propensity to aggregate as are undergoing an autocatalytic activation, evidenced by gel filtration behavior. Gel or modulating factors are lost as activation occurs. This activation is an artificial pheelectrofocusing experiments indicate that the proteins differ in isoelectric point by nomenon and does not operate physiologically since sperm receptor hydrolase and 0.2 pH units; however, separation utilizing preparative electrofocusing has been se- vitelline detaching activities are fully acpresent verely hampered by precipitation of the tive at the esterase concentration in freshly prepared fertilization product. proteins as they approach their isoelectric These hypotheses regarding the activation points.
CARROLL AND EPEL plasma membrane vitelline layerplasma membrane attachment
sperm receptor
w
vitelline layer cortical
granule
FIG. 9. Roles of the proteases released at fertilization from the sea urchin egg. The cortical granules contain two proteases; one acts on vitelline layerplasma membrane attachments and the other on sperm receptors.
mechanism have not been thoroughly investigated but preliminary results indicate a change in isoelectric point following activation (Carroll and Epel, unpublished observation). Our working model for the role of the cortical granule proteases in fertilization is presented in Fig. 9. In this model, the vitelline layer is attached to the egg plasma membrane by a protease-sensitive attachment . Sperm receptors are contained in the vitelline layer as proteins distinct from vitelline layer-plasma membrane attachments. The vitelline layer-plasma membrane attachments could be through noncovalent linkages or peptide bonds. Proteolysis of the latter type of connectives would be sufficient to delaminate vitelline layer and plasma membrane, whereas proteolysis of molecules involved in noncovalent bonding would require an additional conformational change for delamination to be completed. When the fertilizing sperm initiates the cortical reaction, the proteases contained in the cortical granules are released, along with the other colloids known to be present. The protease digests plasma membrane-vitelline layer bonds, attach-
Sea Urchin Egg Protease
31
ment points of bound sperm and any supernumerary sperm receptors. Actual elevation of the vitelline layer requires additional components contained in the cortical granules (Carroll and Epel, 1974). Perhaps the hydration of these cortical granule components supplies the necessary force for elevation. We thank Elizabeth Baker for excellent technical assistance. Dr. E. W. Byrd, Dr. C. K. Franker, Dr. C. K. Mathews, and Dr. M. J. Tegner are thanked for their review of the manuscript. Ionophore A23187 was kindly provided by Dr. R. Hamill of the E. Lilly Co., Indianapolis. This work was supported by research grants from NSF and The Population Council. E.J.C. is a postdoctoral fellow of The Population Council. REFERENCES AKETA, K. (1967). On the sperm-egg bonding as the initial step of fertilization in the sea urchin. Embryologia 9, 238-245. BERG, W. E. (19671. Some experimental techniques for eggs and embryos of marine invertebrates. In “Methods in Developmental Biology,” (F. H. Wilt and N. K. Wessels, eds.), pp. 767-776. Crowell, New York. CARROLL, E. J., JR., and EPEL, D. (1974). Elevation and hardening of the fertilization membrane in sea urchin eggs: Role of the soluble fertilization product. Exp. Cell Res., in press. EDSALL, J. T. (1953). The size, shape and hydration of protein molecules. In “The Proteins,” (H. Neurath and K. Bailey, eds.), Vol. lB, pp. 549-726. Academic Press, New York. GAREN, A., and LEVINTHAL, C. (1960). A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim. Biophys. Acta 38: 470-483. HAGSTRUM, B. E. (1956). Studies on polyspermy in sea urchins. Ark. Zool. 10, 307-315. ISHIHARA, K. (1964). Release of acid polysaccharides following fertilization of sea urchins. Erp. Cell Res. 36, 354-367. L~NNING, S. (1967). Electron microscopic studies of the block to polyspermy. Sarsia 30, 107-116. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MARES-GUIA, M., and SHAW, E. (1965). Studies on the active center of trypsin. The binding of amidines and guanidines as models of the substrate side chain. J. Biol. Chem. 240, 1579-1585.
32
DEVELOPMENTALBIOLOGY
MARTIN, R. G., and AMES, B. H. (1961). A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. Eiol. Chem. 236, 1372-1379. RICK, W. (1963). Trypsin. In “Methods in Enzymatic Analysis (H. U. Bergmyer, ed.), pp. 807-818. Academic Press, New York. ROBINSON, N. C., TYE, R. W., NEURATH, H., and WALSH, K. A. (1971). Isolation of trypsins by affinity chromatography. Biochemistry 10, 2743-2747. SCHUEL, H., WILSON, W. L., BRESSLER,R. S., KELLY, J. W., and WILSON, J. R. (1972). Purification of cortical granules from unfertilized sea urchin egg homogenates by zonal centrifugation. Deuelop. Eiol. 29, 307-320. SCHUEL, H., WILSON, W. L., CHEN, K., and LORAND, L. (1973). A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization. Deuelop. Biol. 34, 175-186. STEINHARDT, R. A., and EPEL, D. (1974). Activation of
VOLUME 44. 1975
sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. USA 71, 1915-1919. VACQUIER, V. D., EPEL, D., and DOUGLAS, L. (1972). Sea urchin eggs release protease activity at fertilization. Nature (London) 237, 34-36, VACQUIER, V. D., and Payne, J. E. (1973). Methods for quantitating sea urchin sperm-egg binding. Exp. Cell Res. 82, 227-235. VACQUIER, V. D., TECNER, M. J., and EPEL, D. (1973a). Protease activity establishes block against polyspermy in sea urchin eggs. Nature (London) 240, 352-353. VAcqunm, V. D., TEGNER, M. J., and EPEL, D. (1973b). Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. 80, 111-119. WARBURG, O., and CHRISTIAN, W. (1941). Isolierung und Kristallisation des glrungsferments Enolase. Biochem. 2. 310, 384-421. WELLNER, D. (1971). Electrofocusing in gels. Anal. Chem. 43,59A-65A.
BIOLOGY
Isolation
44, 22-32 (1975)
and Biological Sea Urchin EDWARD
Scripps Institution
of Oceanography,
Activity
of the Proteases
Eggs following
J. CARROLL,
JR.
Fertilization
AND DAVID
Marine Biology Research Division, La Jolla, California 92037 Accepted
Released by
EPEL Uniuersity
of California,
San Diego,
December 23, 1974
The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity on p-aminobenzamidine-Sepharose columns. Elution profiles from affinity chromatography columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low PH. The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between lo5 and lOa were demonstrated by gel filtration. INTRODUCTION
Sea urchin eggs release macromolecules into the surrounding seawater following fertilization. This solution of macromolecules is referred to as the fertilization product (Ishihara, 1964) and is primarily composed of the soluble cortical granule exudate. The exudate contains protease activity which is sensitive to soybean trypsin inhibitor’ (Vacquier et al., 1972). The function of this activity has been investigated by fertilizing eggs in the presence of STI. Two processes are prevented: (1) The elevation of the fertilization membrane (Hagstrom, 1956; Liinning, 1967; Vacquier et al., 1972; Schuel et al., 1973) and (2) the detachment of excess sperm ‘The abbreviation inhibitor.
used is: STI, soybean trypsin
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
(Vacquier et al., 1973a). Such eggs also become polyspermic (Vacquier et al., 1972; Schuel et al., 1973), suggesting that the protease activity is also involved in the block against polyspermy. The role of this enzyme activity has also been investigated by incubating eggs in the fertilization product. This treatment modifies the egg surface and also prevents subsequent fertilization (Vacquier et al., 1973a). This loss of fertilizability (fertilization-inhibitor activity) is associated with an inability of sperm to attach to the vitelline layer, most likely from alteration of proteinaceous sperm receptors on this layer (Aketa, 1967). Because both fertilization-inhibitor activity and protease activity are prevented by ST1 and because both have similar heat inactivation curves, it was postulated that both activities were properties of the same pro-
CARROLL AND EPEL
tein (Vacquier et al., 1973b). In this paper we report our progress on the purification of the protease activity of the fertilization product. Polyacylamidegel electrofocusing revealed two proteases in the fertilization product. These proteases, partially resolved into two fractions by affinity chromatography, had dramatically different biological activities. One fraction alters the vitelline layer such that sperm cannot bind to and fertilize eggs; we shall refer to this activity as “sperm receptor hydrolase”. The other protease fraction’ is involved in vitelline layer elevation through cleaving connections between the vitelline layer and egg surface; we shall refer to this activity as “vitelline delaminase activity”. MATERIALS
AND
METHODS
Preparation of gametes. Shedding of gametes was induced in the sea urchin Strongylocentrotus purpuratus by injection of 0.5 A4 KC1 into the coelomoic cavity. Sperm were collected “dry” and stored at 0°C. Egg jelly was removed by washing eggs for 3 min in seawater which was acidified to pH 5.0 with 1 N HCl or by sieving egg suspensions five times through No. 183 Nitex mesh. The extent of dejellying was ascertained by gentle hand centrifugation of a portion of the egg suspension. The presence of a homogenous pellet was taken as the endpoint. Quantitation of egg and sperm concentrations was done according to Vacquier and Payne (1974). Bioaways. Sperm receptor hydrolase activity was assayed using inhibition of fertilization as the criterion, as described by Vacquier et al. (1973a) with minor modifications. The egg concentration used was 500/ml. Just before use, sperm suspensions were diluted in seawater from “dry” semen to an absorbance at 340 nm of 1.0 (approximately 1.20 x lo8 sperm/ml). To 200 ~1 of egg suspension was added 50 ~1 of the fraction to be tested; the suspension was incubated for 30 min at 16°C. Following
Sea Urchin
Egg Protease
23
this incubation, 50 ~1 of sperm suspension was added and the extent of fertiliz; ation determined by scoring for the presence of hyaline layers 30 min postinsemination and for cleavage at 90 min post-insemination. The assay of vitelline delaminase activity was as described by Carroll and Epel (1974). The presence of activity is indicated by normal elevation of a fertilization membrane when eggs are activated in the presence of STI. A small number (ca 100) of dejellied S. purpuratus eggs are incubated with the fraction to be tested in a covered Syracuse dish at 16°C. At the conclusion of the incubation, ST1 and the parthenogenetic agent, ionophore A23187 (Steinhardt and Epel, 1974), were added to final concentrations of 0.37 mg/ml and 5 PM, respectively. Controls of no fraction and solvent (dimethyl sulfoxide) were run concurrently. The eggs were then immediately scored for the presence of elevated vitelline layers. Preparation of fertilization product. The method of Vacquier et al. (1973a) was used, with minor modification. Fertilization was accomplished by mixing a 50% (V/v) egg suspension with an equal volume of sperm suspension containing 3 ml of “dry” semen for each 25 ml of packed egg cell volume. All manipulations of fertilization product preparations and purified fractions were done in 0.01-0.10 M sodium acetate, pH 4.0, to prevent “activation”, as described in Results. Manipulations of preparations requiring exposure to neutral or alkaline pH were accomplished as quickly as possible. Enzyme assays. Esterase activity was measured according to Rick (1963) except that reaction mixtures contained 60 mM Tris-HCl, 0.5 mM a-N-benzoyl-r.-arginine ethyl ester at pH 8.0. Assays were run at ambient temperature. The Ki of the esterase activity for p-aminobenzamidine was determined from Lineweaver-Burk plots of data obtained from assays at ten substrate
24
DEVELOPMENTALBIOLOGY
concentrations in the presence and absence of 1.36 x lo-‘M p-aminobenzamidine. Sucrose gradient centrifugation. This was accomplished as described by Martin and Ames (1961) using 5-20s linear sucrose gradients containing 0.1 M Tris-HCl0.4 M NaCl, pH 8.0. Standards were Escherichia coli alkaline phosphatase and bovine hemoglobin. Sedimentation rates were calculated using the published sedimentation coefficients for alkaline phosphatase (Garen and Levinthal, 1960) and bovine hemoglobin (Edsall, 1953). Alkaline phosphatase activity was assayed by incubating aliquots of fractions in 1.0 ml of 1.0 mM p-nitrophenyl phosphate-O.1 M Tris-HCl at pH 8.0 and 37°C for 45 min. The reaction mixture was diluted to 3 ml with water and read at 412 nm in a Gilford Model 300N rapid sampler calorimeter. Electrofocusing. Analytical electrofocusing was essentially according to Wellner (1971) using final concentrations of 4.8% acrylamide, 0.25% N,N,N’,N’-tetramethylethylenediamine, and l-1.4% pH 3-5 ampholines plus 0.6% pH 3-10 ampholines. The anodic solution contained 0.96% H,PO, and the cathodic solution contained 1.7% ethylenediamine.
VOLUME 44, 1975
justed to 4.9 with NaOH. The coupling reaction was initiated by the addition of 1.7 g of 1-cyclohexyl-3-(2-morpholino-ethyl)carbodiimide metho-p-toluene-sulfonate. The pH was maintained at 4.6-4.9 for 5.5 hr at which time the gel was exhaustively washed with water and resuspended in 0.1 M Tris-HCl-0.4 M NaCl, pH 8.0. Protein concentrations were estimated according to either Lowry et al. (1951) or Warburg and Christian (1941). All reagents were prepared from commercially available products with deionized, glass-distilled water. RESULTS
Purification tilization
and Biological Activity of FerProduct E&erase Actiuity
Isoelectric precipitation. The first step in purification was isolation of the enzyme from seawater. When preparations of fertilization product were adjusted to pH 4.0, a precipitate formed which was enriched in esterase activity; a typical preparation is presented in Table 1. The esterase activity was found only in the pH 4.0-insoluble fraction. In various preparations the specific activity was increased 1.3- to 4-fold with an average recovery of 106% (seven Preparation of p-aminobenzamidinepreparations). Assays for sperm receptor CH-Sepharose 4B. Four grams of CH- hydrolase activity indicated that this acSepharose 4B (Pharmacia) were hydrated tivity was also located in the pH 4.0-insoluand washed exhaustively in 0.5 M NaCl. ble fraction. The pH 4.0-supernatant soluThe gel was adjusted to 10% (V/V) in the tion had no detectable effect on fertilizasame medium and 160 mg of p-aminobenz- tion. amidine .2HCl added and the pH adSince cortical granules also contain pTABLE
1
ISOELECTRICPRECIPITATION OF ESTERASE ACTIVITY IN FERTILIZATION PRODUCT Fraction
Crude fertilization
product*
pH 4.0-insoluble fraction’ pH 4.0~soluble fraction
IU”
Mg
IU/mg
8.4
345
0.0243
-
7.2 0.24
224 121
0.0321 0.002
85.7 2.9
Total recovery (percent)
“The esterase assay as described in Materials and Methods was used. * Fertilization product was prepared as described in Materials and Methods. c Precipitation at pH 4.0 was accomplished as described in Results.
Percent of activity in fraction
Purification (n-fold) -
97 3
1.3 -
CARROLLANDEPEL
glucanase activity (Schuel et al ., 1972)) the fractions obtained by isoelectric precipitation were examined for the presence of this activity. The pH 4.0-supernatant fraction contained 96% of the /3-glucanase activity recovered, the remaining 4% was contained in the pH 4.0-precipitate fraction. Affinity chromatography. Initial attempts at affinity chromatography made use of Sepharose resin prepared according to Robinson et al. (1971) with ST1 substituted for ovomucoid. Protease activity of the unfractionated fertilization product or the pH 4.0-insoluble fraction was firmly retained on a column of this resin. However, elution with a pH change (pH 10.5) or ru-N-benzoyl-L-arginine ethyl ester containing solutions (0.05-0.10 M at pH 8.0) was not successful; thus STI-Sepharose was unsuitable as an affinity adsorbent. The competitive trypsin inhibitor paminobenzamidine (Mares-Guia and Shaw, 1965) was tested as a possible ligand for an affinity resin. With the pH 4.0insoluble fraction (dissolved in 0.1 M Tris-HCl, pH 8.0) the K, of p-aminobenzamidine, using a-N-benzoyl-L-arginine ethyl ester as substrate, was 1.6 x 1O-4M. Thus p-aminobenzamidine was a sufficiently good inhibitor to make affinity chromatographic methods feasible. The various batches of p-aminobenzamidineCH-Sepharose 4B resin that were prepared did not adsorb detectable quantities of either commercial chymotrypsin or trypsinogen (based on absorbance at 280 nm). Commercial trypsin was tightly bound to the extent of 3-7 mg of trypsin/ml of resin (based on absorbance at 280 nm) and could be specifically eluted with cY-N-benzoyl-Larginine ethyl ester or p-aminobenzamidine-containing solutions (0.1-20 mM, pH 8.0). Elution of trypsin was also accomplished using either acidic or basic pH changes (pH 3.0 or 11.6). The esterase activity of the pH 4.0insoluble fraction was also bound by this matrix. Attempts at elution of bound pro-
Sea Urchin Egg Protease
25
tein using p-aminobenzamidine (1 .Oto 100 mM, pH 8.0) or cu-N-benzoyl-L-arginine ethyl ester-containing solutions (1.0-250 mM, pH 8.0) met with limited success, viz, the eluted protein was dilute and recovered in poor yield. Elution of bound enzyme was best accomplished using a basic pH change. A preparation of fertilization product purified by isoelectric precipitation and containing 3.7 international units (IU) of esterase activity and 24 mg of protein (in 0.1 M Tris-HCl-0.4 M NaCl, pH 8.0) was applied to a p-aminobenzamidineSepharose column at a flow rate of 450 ml/hr. Under these conditions, 35% of the applied esterase activity was bound to the column. An elution diagram of the esterase activity and protein eluted with 0.1 M NH,OH-0.4 M NaCl (pH approximately 11.6) is shown in Fig. 1. The esterase activity and protein peaks are incongruent, meaning that the specific activity is not constant across the profile and suggesting the existence or presence of more than one enzyme. The leading and trailing edges of the peak had limiting specific activity values of 0.01 and 0.10 IU/mg, respectively. The degree of separation of protein and esterase activity could be considerably improved by decreasing the flow rate, as shown in Fig. 2. The conditions and enzyme preparation used were virtually identical with those of Fig. 1 except that the flow rate was 60 ml/hr. Using this flow rate, 74% of the esterase activity applied was bound to the column, and in other experiments close to 100%of the esterase activity was bound. Also under these latter conditions almost all of the protein of the pH 4.0-precipitate fraction was bound to the column, indicating that this fraction is composed almost entirely of protease activity. The effect of column dimension was not thoroughly investigated, but the greatest degree of separation was observed when columns had length/diameter ratios of approximately 20.
26
DEVELOPMENTALBIOLOGY
VOLUME 44, 1975
7 6
-10 7 0* -4z 3
-6,”
-2 -
Fraction Number FIG. 1. Chromatography of the pH 4.0-insoluble fraction of fertilization product bound to a p-aminobenzamidine-CH-Sepharose 4B column. A 2.2 x 4-cm column was poured and equilibrated with 0.1 M Tris-HCI-0.4 M NaCl, pH 8.0. A solution containing 3.7 IU of esterase activity and 24 mg of protein was applied to the column in the same buffer. The column was washed with about 15 bed-volumes of the same buffer until the absorbance of the eluate was 10.02. The bound protein was eluted at a flow rate of 450 ml/hr with a 0.1 M NH,OH-0.4 M NaCl solution; the eluate was collected in tubes containing sufficient 1.0 M sodium acetate to maintain the pH at 4.0.0, mg/ml protein; 0, IU/ml esterase activity; x, IU/mg.
0
2
L
, ,
iIO
20
-*---y--.
30 Fraction Number
40
50
FIG. 2. Chromatography of the pH 4.0-insoluble fraction of fertilization product bound to a p-aminobenzamidine-CH-Sepharose 4B column. A 2.2 x 4-cm column was poured, equilibrated, and developed as described in the legend to Fig. 1, except that the flow rate was 60 ml/hr. A solution containing 5.5 IU of esterase activity and 36 mg of protein was applied to the column. 0, mg/ml protein; 0, III/ml esterase activity; x, W/ml.
CARROLL AND EPEL
Polyacrylamide-gel electrofocusing provided direct evidence for the presence of multiple proteins in the preparations purified by isoelectric precipitation and affinity chromatography. Using the combinedactivity fraction from the affinity column (indicated by the bar in Fig. 2), two proteins were resolved with isoelectric points of 4.7 and 4.9 (Fig. 3). Extraction and enzymatic assay of the separated proteins from the gel was not possible since the proteins precipitate at equilibrium and gel extracts contained an insufficient amount of protein for assay. Use of higher levels of input protein diminished resolution, but all enzymatic activity extracted was coincident with protein (Fig. 4). As noted, two functions attributed to the fertilization product protease activity relate to detachment of the vitelline layer and destruction of sperm receptors. As shown, there are at least two. species of protease in the fertilization product. Although complete separation of the proteases was not accomplished on a preparative basis, we made use of the widely differing specific activities across the affinity chromatogram peak (Figs. 1 and 2). Collection of the leading and trailing edges yielded partial separation into a lowspecific-activity fraction and a high-specific-activity fraction. Equivalent amounts of activity (a-N-benzoyl-L-arginine ethyl ester as substrate) were then applied to eggs in bioassay procedures. The results of a typical bioassay are presented in Fig. 5. In the vitelline detaching assay, a positive result is indicated by the presence of normally elevated fertilization membranes upon parthenogenetic activation of the test eggs with ionophore A23187 in the presence of STI. In the absence of this activity (Fig. 5A) the fertilization membrane remains firmly attached to the egg surface at many sites and has a rosette appearance. A positive result in the sperm receptor hydrolase assay is indicated
27
Sea Urchin Egg Protease
I
I
1
I
1
I
0.8 1.0 0.2 0.4 0.6 Fraction of Gel Length FIG. 3. Polyacrylamide-gel electrofocusing of the pH 4.0.insoluble fraction of fertilization product purified by affinity chromatography. Electrofocusing was at 55 V for 17 hr at 4°C; 94 pg of protein were applied. The preparation used was the pooled fractions indicated by the bar in Fig. 2. A parallel gel was frozen on a paraffin block and sliced into 58 fractions; 1 ml of water was added to each slice and the resultant pH measured after 24 hr of extraction.
I
+
FIG. 4. Polyacrylamide-gel electrofocusing and enzymatic activity assay of the pH 4.0~insoluble fraction of fertilization product purified by affinity chromatography. Conditions for electrofocusing and pH determination were as described in the legend to Fig. 3 and in Materials and Methods. Three milligrams of the preparation described in the legend to Fig. 3 were applied to the gel. The schematic representation of the gel was obtained from measurements of the precipitated protein visible in the gel at the end of the run. The gel was then sliced into eight fractions and homogenized in a Dounce apparatus with 0.5 ml of 1.0 M Tris-HCl, pH 8.0, at O”C, per mm of gel slice. Extraction was for 90 min; then the homogenates were centrifuged at 10,000 g for 1 min and the extracts were assayed for esterase activity.
FIG. 5. Biological assays for vitelline detaching activity and sperm receptor hydrolase activity in the lowand high-specific-activity fractions from an-aminobenzamidine-Sepharose-CH 4B column. In a total volume of 250 ~1, 100 eggs were incubated for 30 min at 16°C in each of the various fractions. Assays using the low-specific-activity fraction contained 44 Fg of enzyme at 0.028 IU/mg; assays containing the high-specificactivity fraction contained 12 fig of enzyme at 0.093 IU/mg. Assays using the mixture of fractions contained the same amounts of the enzymes as above. Vitelline detaching assay: Eggs treated with seawater (A), low-specific-activity fraction (B), high-specific-activity fraction (C), a’ mixture of low- and high-specific-activity fractions activated with ionophore A23187 in the presence of ST1 (D). Sperm receptor hydrolase assay: Eggs treated with seawater (E), low-specific-activity fraction (F), high-specific activity fraction (G), a mixture of low- and high-specific-activity fractions and fertilized (H).
by the failure of sperm to bind and fertilize test eggs; in the absence of this activity eggs fertilized normally (Fig. 5E). The low-specific-activity fraction had vitelline detaching activity (Fig. 5B) but no sperm receptor hydrolase activity (Fig. 5F). Conversely, the high-specific-activity fraction had no vitelline detaching activity (Fig. 5C) but had sperm receptor hydrolase activity (Fig. 5G). These two activities are independent, as a mixture of the two fractions exhibited both activities (Figs. 5D and H). These data suggest that these biological activities reside in at least two separate protease molecules. Additional Properties of Fertilization uct E&erase Activity
Prod-
Activation of fertilization product esterase activity. Esterase activity of the fertilization product increases five- to tenfold during storage at 0°C (Vacquier, unpublished observation). This activation becomes apparent by 24 hr and continues for several days. There is concomitent loss of
sperm receptor hydrolase activity as determined by the bioassay. We have also noted that these activated preparations digest the vitelline layer. As sperm receptor hydrolase activity cannot be assayed when the vitelline layer is absent, we do not know if activation directly alters sperm receptor hydrolase activity. Both esterase activity and sperm receptor hydrolase activity remained constant when preparations were stored at O”C, pH 4.5, or at -2O”C, pH 8.4 or pH 4.5, whereas storage at O”C, pH 8.4, resulted in approximately sevenfold activation over a 4-day period (Fig. 6). Accordingly, fertilization product preparations were routinely stored at - 20°C as a suspension in 0.05 M sodium acetate-50% glycerol, pH 4.0-4.5. Preparations were stored in the liquid state (50% glycerol) because rapid dissolution of the precipitate was possible following readjustment of the pH to 8.0. When these precipitates were frozen, dissolution at pH 8.0 proceeded very slowly and activation frequently occurred.
CARROLL AND EPEL
8
16 DAYS
Sea Urchin
24
FIG. 6. Activation of fertilization product esterase activity. Portions of a fertilization product preparation were stored under various conditions and assayed at various times. To one portion was added %Ovolume of 0.5 M sodium acetate, pH 4.0, and to another was pH 8.0. Oneadded SO volume of 0.5 M Tris-HCI, milliter aliquots were stored at 0” or at -22°C and assayed after the indicated time of storage. The final pH’s were 4.45 and 8.35, respectively. q , pH 8.35, -22°C; 0, pH 8.35, 0°C; n , pH 4.45, -22°C; l , pH 4.45. 0°C.
Gel filtration. When the pH 4.0-insoluble fraction was chromatographed on a Sepharose 4B column, multiple peaks of e&erase activity were apparent with approximate molecular weights between lo5 and lo6 (Fig. 7). This heterogeneity of esterase activity is possibly the result of aggregation, since rechromatography of fraction I resulted in the appearance of fractions I and II. The same elution patterns were obtained with several preparations both at pH 5.6 and 8.0. Molecular weight estimation. From velocity sedimentation in sucrose, the single peak of esterase activity was 4.5 S (Fig. 8). This corresponds to a molecular weight of 47,000.
Egg Protease
29
which alters the vitelline layer such that sperm cannot bind to it and cannot fertilize the egg. Therefore, the vitelline layer is a physical barrier to fertilization of the egg, and the fertilizing sperm cell must bind and interact with a specific receptor on the vitelline layer surface prior to penetration of the egg. The high-specific-activity fraction also detaches bound sperm from eggs (Carroll and Epel, unpublished observations) . Destruction of sperm receptors on the vitelline layer and detachment of supernumerary sperm from the vitelline layer are important components in the block against polyspermy (Vacquier et al., 1973a). Aketa (1967) has shown that the vitelline layer contains sperm-binding proteins. Proteolytic action on these molecules by sperm receptor hydrolase before sperm binding would prevent a sperm from binding to the receptor whereas proteolysis of a receptor after binding would result in sperm detachment. Previous work has shown that sperm detachment and fertilization membrane elevation occur simultaneously (Vacquier and Payne, 1974). Since vitelline detaching activity and sperm receptor hydrolase ac-
I 16 P 12
4,;
08
30 : 3 20 -
04
IO
Na
DISCUSSION
In this paper we show that the protease fraction of the fertilization product consists of at least two enzymes. A low-specificactivity fraction contains vitelline detaching activity which apparently removes connections between the plasma membrane and vitelline layer as a part of the elevation process. The high-specific-activity fraction contains sperm receptor hydrolase activity
15
20 25 30 FRACTION NUMBER
35
FIG. 7. Chromatography of the pH 4.0-insoluble fraction of fertilization product on a Sepharose 4B column. A 1.4 x 36-cm column was poured and equilibrated with 0.1 M Tris-citrate at pH 5.6 and 4°C. A solution containing 1.24 IU of esterase activity was applied in the same buffer containing 8% sucrose. The void volume was determined in a separate run with blue dextran as marker. 0, absorbance at 280 nm; 0, IU/ml, esterase activity.
30
DEVELOPMENTAL
VOLUME 44, 1975
BIOLOGY
BOTTOM
TOP
014 -012
006
%
002 ‘0 ‘a
lo-
20 Fracl~on Number
o
30
-
-o--o-o
40
FIG. 8. Sucrose gradient ultracentrifugation of the pH 4.0~insoluble fraction of fertilization product. Two-hundred-forty microliters of a solution containing 0.085 IU of esterase activity (1.3 mg of protein), 2 mg of hemoglobin and 25 rg of E. coli alkaline phosphatase were layered on a 5.4-ml gradient. Centrifugation was for 45 hr at 49,500 rpm in a SW5OL rotor at 4°C. At the end of the run fractions were collected, 10 pl were assayed for alkaline phosphatase activity (0) and 50 ~1 assayed for e&erase activity (O), and 1.5 ml of water was added to the remaining solution and the absorbance at 415 nm was monitored for hemoglobin (A).
tivity apparently reside in different mole‘The proteases of the fertilization product cules, the simultaneous release of two pro- are highly specific enzymes. Bovine trypsin teases from the cortical granules now ex- and various other proteases (Berg, 1967) plains the coincidence of elevation and will digest the vitelline layer such that it sperm detachment. Attachment of the does not elevate at fertilization. The trypplasma membrane to the vitelline layer by sin-like enzymes of the fertilization product, however, do not act in this fashion. protease-sensitive sites is not a prerequisite layer such that for fertilization since delaminated eggs are One alters the vitelline fully fertilizable. These results also suggest sperm do not bind to it, and another that plasma membrane-vitelline layer con- digests points of attachment between the nections are distinct from the sites of plasma membrane and vitelline layer as a part of the elevation process. However, sperm binding and penetration. The complete separation of the two pro- when the esterase activity of the fertilizateases has not yet been realized, and this tion product activates during storage at pH task is a formidable one for several reasons. 8, specificity is lost and its action on the The biological activities are unstable at pH vitelline layer becomes like that of bovine 8.0; activation of esterase activity occurs trypsin in digesting the vitelline layer. The mechanism of this activation is with a concomitant loss in specificity. The presently unclear. Possibly the enzymes enzymes have similar molecular weights and have a propensity to aggregate as are undergoing an autocatalytic activation, evidenced by gel filtration behavior. Gel or modulating factors are lost as activation occurs. This activation is an artificial pheelectrofocusing experiments indicate that the proteins differ in isoelectric point by nomenon and does not operate physiologically since sperm receptor hydrolase and 0.2 pH units; however, separation utilizing preparative electrofocusing has been se- vitelline detaching activities are fully acpresent verely hampered by precipitation of the tive at the esterase concentration in freshly prepared fertilization product. proteins as they approach their isoelectric These hypotheses regarding the activation points.
CARROLL AND EPEL plasma membrane vitelline layerplasma membrane attachment
sperm receptor
w
vitelline layer cortical
granule
FIG. 9. Roles of the proteases released at fertilization from the sea urchin egg. The cortical granules contain two proteases; one acts on vitelline layerplasma membrane attachments and the other on sperm receptors.
mechanism have not been thoroughly investigated but preliminary results indicate a change in isoelectric point following activation (Carroll and Epel, unpublished observation). Our working model for the role of the cortical granule proteases in fertilization is presented in Fig. 9. In this model, the vitelline layer is attached to the egg plasma membrane by a protease-sensitive attachment . Sperm receptors are contained in the vitelline layer as proteins distinct from vitelline layer-plasma membrane attachments. The vitelline layer-plasma membrane attachments could be through noncovalent linkages or peptide bonds. Proteolysis of the latter type of connectives would be sufficient to delaminate vitelline layer and plasma membrane, whereas proteolysis of molecules involved in noncovalent bonding would require an additional conformational change for delamination to be completed. When the fertilizing sperm initiates the cortical reaction, the proteases contained in the cortical granules are released, along with the other colloids known to be present. The protease digests plasma membrane-vitelline layer bonds, attach-
Sea Urchin Egg Protease
31
ment points of bound sperm and any supernumerary sperm receptors. Actual elevation of the vitelline layer requires additional components contained in the cortical granules (Carroll and Epel, 1974). Perhaps the hydration of these cortical granule components supplies the necessary force for elevation. We thank Elizabeth Baker for excellent technical assistance. Dr. E. W. Byrd, Dr. C. K. Franker, Dr. C. K. Mathews, and Dr. M. J. Tegner are thanked for their review of the manuscript. Ionophore A23187 was kindly provided by Dr. R. Hamill of the E. Lilly Co., Indianapolis. This work was supported by research grants from NSF and The Population Council. E.J.C. is a postdoctoral fellow of The Population Council. REFERENCES AKETA, K. (1967). On the sperm-egg bonding as the initial step of fertilization in the sea urchin. Embryologia 9, 238-245. BERG, W. E. (19671. Some experimental techniques for eggs and embryos of marine invertebrates. In “Methods in Developmental Biology,” (F. H. Wilt and N. K. Wessels, eds.), pp. 767-776. Crowell, New York. CARROLL, E. J., JR., and EPEL, D. (1974). Elevation and hardening of the fertilization membrane in sea urchin eggs: Role of the soluble fertilization product. Exp. Cell Res., in press. EDSALL, J. T. (1953). The size, shape and hydration of protein molecules. In “The Proteins,” (H. Neurath and K. Bailey, eds.), Vol. lB, pp. 549-726. Academic Press, New York. GAREN, A., and LEVINTHAL, C. (1960). A fine-structure genetic and chemical study of the enzyme alkaline phosphatase of E. coli. I. Purification and characterization of alkaline phosphatase. Biochim. Biophys. Acta 38: 470-483. HAGSTRUM, B. E. (1956). Studies on polyspermy in sea urchins. Ark. Zool. 10, 307-315. ISHIHARA, K. (1964). Release of acid polysaccharides following fertilization of sea urchins. Erp. Cell Res. 36, 354-367. L~NNING, S. (1967). Electron microscopic studies of the block to polyspermy. Sarsia 30, 107-116. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MARES-GUIA, M., and SHAW, E. (1965). Studies on the active center of trypsin. The binding of amidines and guanidines as models of the substrate side chain. J. Biol. Chem. 240, 1579-1585.
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sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. USA 71, 1915-1919. VACQUIER, V. D., EPEL, D., and DOUGLAS, L. (1972). Sea urchin eggs release protease activity at fertilization. Nature (London) 237, 34-36, VACQUIER, V. D., and Payne, J. E. (1973). Methods for quantitating sea urchin sperm-egg binding. Exp. Cell Res. 82, 227-235. VACQUIER, V. D., TECNER, M. J., and EPEL, D. (1973a). Protease activity establishes block against polyspermy in sea urchin eggs. Nature (London) 240, 352-353. VAcqunm, V. D., TEGNER, M. J., and EPEL, D. (1973b). Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy. Exp. Cell Res. 80, 111-119. WARBURG, O., and CHRISTIAN, W. (1941). Isolierung und Kristallisation des glrungsferments Enolase. Biochem. 2. 310, 384-421. WELLNER, D. (1971). Electrofocusing in gels. Anal. Chem. 43,59A-65A.
