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[28] Cortical Granule Proteases from Sea Urchin Eggs B y EDWARD J. CARROLL, JR.
Sea urchin eggs contain protease activity in the cortical granules, a set of secretory organelles adjacent to the plasma membrane. 1,2 After the fusion of cortical granule and plasma membranes at fertilization, the contents of the cortical granules are extruded into the space between the plasma membrane and the overlying vitelline layer. The vitelline layer is semipermeable, as part of the protease activity is found in the supernatant seawater. This protease activity consists of at least two enzymes which have two separable functions in vivo2 One enzyme, termed sperm receptor hydrolase, modifies the sperm-binding properties of eggs and is important in removing supernumerary sperm-binding sites and spermatozoa as a part of the block against polyspermy. Another enzyme, vitelline delaminase, functions in the first step in elevation of the extracellular vitelline layer by cleaving vitelline layer-plasma membrane attachments. Actual elevation of this layer requires additional cortical granule components. 4
Assay Methods The protease activity of the sea urchin egg cortical granule exudate can be determined qualitatively using two bioassays or quantitatively using a radioactive protein or synthetic ester substrate assay.
Bioassays As noted, two biological activities attributed to the egg cortical granule exudate relate to detachment of the vitelline layer (vitelline delaminase) and destruction of sperm receptors (sperm receptor hydrolase). Vitelline Delaminase Principle. Eggs incubated in solutions containing vitelline delaminase activity will elevate a fertilization membrane in the presence of soybean trypsin inhibitor when activated with butyric acid or the divalent cation
iV. D. Vacquier, D. Epel, and L. A. Douglas, Nature (London) 237, 34 (1972). H. Schuel, W. L. Wilson, R. S. Bressler, J. W. Kelly, and J. R. Wilson, Dev. Biol. 29, 307 (1973). E. J. Carroll, Jr., and D. Epel, Dev. Biol. 44, 22 (1975). ~E. J. Carroll, Jr., and D. Epel, Exp. Cell Res. 90, 429 (1975).
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P a O T E A S E S FROM GAMETES AND D E V E L O P I N G EMBRYOS
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ionophore A23187. 4 Control eggs incubated in the absence of this activity exhibit a fertilization membrane that is "blebbed" or has a rosette appearance. Thus this protease activity of the cortical granule exudate functions in modifying connections in the fertilizing egg cell between the plasma membrane and vitelline layer such that normal elevation of the vitelline layer can occur. The ionophore procedure of egg activation is detailed here, as it is the easiest, and most reproducible, method; the butyric acid method of egg activation has been described2
Reagents Ionophore A23187 (kindly provided by Dr. R. Hamill of the E. Lilly Co., Indianapolis): 5 mM in dimethyl sulfoxide. Always store in a dark, stoppered bottle covered with foil and refrigerate when not in use. Soybean trypsin inhibitor solution: 3.7 mg/ml in seawater (Sigma, chromatographically purified)
Procedure. A stock egg suspension is prepared containing approximately 500 cells/ml. For the assay, 200 ~l of this suspension are pipetted (wide-bore pipette) into 27-mm Syracuse watch glasses (A. H. Thomas Co.) together with 50 ~l of seawater or test solution (in seawater). The watch glasses are placed in a covered petri dish in a water bath at 16 °. After incubation for 15-60 min, 25 ~l of soybean trypsin inhibitor solution and 5 ~l of ionophore solution are added with rapid mixing. The cells are then examined at X400 magnification and scored for the presence of elevated fertilization membranes. Sperm Receptor Hydrolase Principle. After incubation of unfertilized eggs in solutions containing this activity, they will not bind sperm, nor will they fertilize. This latter result forms the basis for a bioassay of sperm receptor hydrolase activity2 Reagents. No special reagents are required. Procedure. A stock egg suspension containing approximately 500 cells/ml is prepared. For each assay, 200 ~l of this suspension are pipetted (wide-bore pipette) into 27-mm Syracuse watch glasses. Then 50 ~l of test solution (in seawater) or seawater are added; watch glasses are placed in a covered petri plate and then placed in a water bath at 16 °. After incubation for 15-60 min, the eggs are inseminated with 50 ~l of a sperm suspension prepared by diluting "dry" semen to an absorbance ~J. Bryan, J. Cell Biol. 45, 606 (1970). 6V. D. Vacquier, M. J. Tegner, and D. Epel, Exp. Cell Res. 80, 111 (1973).
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at 340 nm of 1.0. This sperm suspension contains approximately 1.2 X 10s cells/ml. 7 To score percentage of fertilization, the eggs are examined at 20 rain for the presence of hyaline layers and elevated fertilization membranes and again at 90 rain after insemination for cleavage. Chemical Assays
Protease Activity Principle. The extent of proteolysis of radiolabeled sea urchin blastula protein is monitored by determination of the radioactivity solubilized into trichloroacetic acid (TCA). 1
Reagents Buffer: 0.1 M Tris-HC1, pH 7.8 Substrate. A 2-liter 24-hr culture (16°-18 °) of fertilized Strongylocentrotus purpuratus or Lytechinus pictus embryos (0.15%, v/v) is reduced to 1 liter. For each 100 ml of this concentrated culture, add 300 t~l of a 100 ~Ci/ml solution of [3H]L-valine. The culture is further incubated for 1-4 hr. The embryos are pelleted by gentle hand centrifugation, and the supernatant seawater is removed by aspiration. The embryos are then extracted in 10% TCA. The resulting precipitate is collected by centrifugation and washed 2 times in 10% TCA by resuspension and centrifugation. A third washing is done in 10% TCA at 90 ° for 20 rain. The TCA-insoluble fraction is then extracted 4 times with chloroform/methanol (1:3 v/v) followed by 3 washes in 95% ethanol. The precipitate fraction is then dissolved in a minimal volume of 0.05 N NaOH and dialyzed against 2 liters of 0.1 M Tris-HC1, pH 7.8, overnight at 4 °. The insoluble protein is removed by centrifugation at 20,000 g for 30 min. Typically, specific radioactivities are on the order of 2 X 107 dpm/mg protein. Approximately 15 mg of labeled protein per 100 ml of 0.15% culture are typically obtained using this procedure. An unlabeled preparation of sea urchin embryo protein is also processed to serve as an unlabeled diluent of the radioactive protein. Carrier protein solution: 50 mg of bovine serum albumin per milliliter in water Trichloroacetic acid (TCA), 10% (v/v)
Procedure. Dilute the 3H-labeled protein to 2.0 mg/ml in 0.1 M TrisHC1, pH 7.8. Add 50 ~l of enzyme solution to 100 t~l of 8H-labeled pro' V . D. Vacquier and J. E. Payne, Exp. Cell Res. 82, 227 (1973).
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P R O T E A S E S FROM GAMETES AND DEVELOPING EMBRYOS
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tein and 50 tLl of 0.l M Tris-HC1, pH 7.8, in a 12-ml conical centrifuge tube at 25 ° . The assays are incubated for up to 2 hr, at which time 100 t,1 of bovine serum albumin carrier protein solution is added with mixing. One ml of ice cold 10% TCA is immediately added with mixing, and the tubes are chilled on an ice bath for 30 min. A zero time blank is prepared by adding the albumin and TCA before enzyme. The tubes are then centrifuged for 5 min at top speed in a clinical centrifuge; 0.8 ml of the supernatant solution is removed and mixed in a scintillation vial with 10 ml of Aquasol (New England Nuclear Corp.) and counted. The limit of linearity of release of TCA-soluble 3H-labeled peptides and amino acid is a function of enzyme concentration and time. Under these conditions, 50 t~l of undiluted crude cortical granule exudate gives a linear release of 3H-labeled products for at least 2 hr.
Esterase Activity Principle. The hydrolysis of the synthetic ester a-N-benzoyl-L-arginine ethyl ester (BAEE) is accompanied by an increase in absorbance at 253 nm. s Reagents Buffer: 0.1 M Tris-HC1, pH 7.8 Substrate: 10 m M B A E E in water
Procedure. Pipette 1.2 ml of buffer into a 3-ml cuvette. Add 0.7 ml of enzyme solution and initiate the reaction with 100 t,1 of substrate. Monitor the change in absorbance for approximately 10 min and calculate the rate of absorbance change per minute (corrected for nonenzymic hydrolysis) and divide by 1.15 to convert change in absorbance per minute to micromoles per minute (1 unit). Specific activity is expressed as units per milligram. Preparation and Purification
Shedding of Gametes. Mature Strongylocentrotus purpuratus can be obtained through Pacific Bio-Marine, Venice, California. The shedding of gametes is induced by the intracoelomic injection of 0.5 M KC1. Alternatively, Aristotle's lantern is removed with forceps, the body fluids are drained, and a small amount of 0.5 M KC1 is poured into the test. In either case the females are then allowed to shed by inverting the animals 8G. W. Schwert and Y. Takenaka, Biochim. Biophys. Acta 16, 570 (1955).
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and allowing the eggs to fall into beakers filled with seawater. The males are shed "dry," i.e., the test is wrapped with a tissue and the animal is inverted over a dry beaker and allowed to shed at 4 °. Preparation o] Cortical Granule Exudate. The eggs are collected and sieved through No. 180 Nitex mesh to remove debris. The eggs are coated with two extracellular layers, which are removed prior to fertilization to facilitate collection of the cortical granule contents. The jelly coat layer which is outermost, is removed by adjustment of the pH of the egg suspension to 5.0 with 0.1-1.0 N HC1. After 3 rain the pH is readjusted to 8.0 with unneutralized 1.0 M Tris. The eggs are washed several times by aspiration of the supernatant seawater and resuspension in fresh seawater. The vitelline layer is an extracellular protein/glycoprotein coat immediately exterior to the egg plasma membrane. Modification of the vitelline layer is effected by brief treatment of eggs with dithiothreitol2 The dejellied, washed eggs are adjusted to a 50% suspension (v/v) and mixed with an equal volume of 0.02 M dithiothreitol, pH 9.1. After a 3-min incubation, the eggs are transferred to a 20-fold excess of normal seawater and washed at least three times. TM The dejellied, dithiothreitol-treated eggs are adiusted to a 50% (v/v) suspension in seawater containing 10 mM Tris-HC1, pH 8.0, and mixed with an equal volume of sperm suspension for fertilization. The sperm suspension is prepared by adding 6 ml of "dry" sperm up to 100 ml with seawater containing 10 mM Tris-HC1, pH 8.0. Three minutes after insemination, the eggs are removed by gentle hand centrifugation. All further manipulations are performed at 0°-4 ° . The sperm and insoluble components of the exudate are removed by centrifugation at 20,000 g for 20 rain. The supernatant solution is referred to as the crude cortical granule exudate. Isoelectric Precipitation. The protease activity contained in the exudate is quantitatively precipitated by the gradual addition of 0.1 volume 1.0 M sodium acetate (pH 4.0) with stirring. 3 The preparation is incubated for 1 hr to overnight. The precipitate is harvested by centrifugation at 20,000 g for 30 rain, then washed twice by resuspension and homogenization in 0.1 M sodium acetate pH 4.0 (Dounce apparatus) using one-tenth of the original volume of seawater. The pH 4.0-insoluble fraction of the exudate can also be washed by cycles of dissolution at pH 8.0 and reprecipitation at pH 4.0, but this can result in a complete loss of biological activity, as will be detailed later. 9D. Epel, A. Weaver, and D. Mazia, Exp. Cell Res. 71, 69 (1970). I°V. D. Vacquier, M. J. Tegner, and D. Epel, Nature (London) 240, 352 (1972).
348
P R O T E A S E S F R O M G A M E T E S AND D E V E L O P I N G E M B R Y O S
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Affinity Chromatography. An effective affinity adsorbent can easily be prepared using the competitive trypsin inhibitor p-aminobenzamidine as a ligand2 CH-Separose-4B (Pharmacia) is hydrated and washed in 0.5 M NaC1. The gel is adjusted to 10% (v/v), and 40 mg of p-aminobenzamidine.2HC1 is added for each gram of dry gel. The pH is adjusted to 4.9 with 1.0 N NaOH and the coupling reaction is initiated by the addition of 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-ptoluene-sulfonate (425 mg per gram of dry gel). The pH of the gel suspension is maintained at 4.6-4.9 for 6 hr with very gentle stirring; the gel is then washed with 0.1 M Tris-HC1-0.4 M NaC1, pH 8.0. The capacity of this resin for bovine trypsin is approximately 7 mg per milliliter of resin. A 2.2 }( 4 cm column of p-aminobenzamidine-Sepharose is poured and equilibrated with 0.1 M Tris-HC1-0.4 M NaC1, pH 8.0, by washing the resin bed with several volumes of buffer. A portion of a protease suspension in pH 4.0 sodium acetate is centrifuged at 20,000 g for 20 min to collect the precipitate. Resuspend the precipitate to a protein concentration of 2.6 mg/ml in water using a Dounce homogenizer. When the precipitate is finely suspended, rapidly add 0.1 volume of 1.0 M Tris-HC1-4.0 M NaC1, pH 8.0, and centrifuge the preparation at 20,000 g for 20 min. Immediately load 15 ml of the enzyme solution onto the column at a flow rate of 60 ml/hr. Wash the column until the absorbance at 280 nm is less than 0.01 when compared to water. Elution of the bound protein is accomplished by applying a 0.1 M NH4OH-0.4 M NaC1 (pH ,-- 11.6) step to the column. Fractions are collected in test tubes containing sufficient 1.0 M sodium acetate, pH 4.0, to buffer the fraction at that pH. Under these conditions 75% of the input esterase activity and protein is bound to the column. Complete binding of all the protein and esterase activity in the pH 4.0 precipitate fraction can be obtained if the amount of resin is increased or the amount of protein applied is decreased. As shown in Fig. 1, the protein fraction eluting with 0.1 M NH4OH0.4 M NaC1 does not elute coincidentally with the esterase activity, suggesting heterogeneity in this fraction. Polyacrylamide gel electrofocusing of the pooled fraction (indicated by the bar in Fig. 1) showed the presence of two proteins of isoelectric points 4.7 and 4.9 (Fig. 2). Complete separation of these two enzymes by affinity chromatography has not yet been successful. The enzymes bind tightly to the resin and seem to elute in an all-or-none fashion. The degree of resolution of the enzymes cluted with a pH change (0.1 M NH4OH-0.4 M NaC1, pit 11.6) is a function of flow rate and column dimension; maximum separation was obtained with a flow rate of 60 ml/hr and a column length:diameter
[28]
SEA URCHIN EGG PROTEASES
349
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FIG. 1. Chromatography of the pH 4.0 precipitate fraction of the cortical granule exudate on p-aminobenzamidine Sepharose. The protein fraction eluting with 0.1 M NH4OH-0.4 M NaC1 is shown. See text for details. Q) O, IU/ml; • •, mg/ml protein; × ×, IU/mg. ratio of 20:1. Although complete separation of the proteases was not achieved using affinity chromatographic techniques, use can be made of the widely differing specific activities across the elution profile (Fig. 1). Collection of the leading and trailing edges yields partial separation into a low specific activity and a high specific activity fraction. Fractions collected in this manner yield biologically monospecific enzymes. The low specific activity fractions contain vitelline delaminase activity, and the sperm receptor hydrolase activity is contained in the high specific activity fractions. Isoelectric Focusing. Preliminary resolution of the two proteases present in cortical granule exudate preparation~ purified by isoelectrie precipitation and affinity chromatography has been obtained using electrofocusing procedures in 4.0 M urea. 1~ The urea is necessary to preclude precipitation of the enzymes at their isoelectric point. SOLUTIONS
Lower electrode solution: 1.85% ethylenediamine Containing 60% sucrose and 4 M urea ~1E. J. Carroll, Jr., unpublished observations (1975).
350
PROTEASES FROM GAMETES AND DEVELOPING EMBRYOS
[28]
/
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/
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l~G. 2. Polyacrylamide gel electrofocusing of the affinity-purified pooled fraction indicated by the bar in Fig. 1. Details of the conditions for electrofocusing, gel slicing, and pH determination were those of E. J. Carroll, Jr., and D. Epel, Dev. Biol. 44, 22 (1975).
Sample solution: 1% pH 3-10 Ampholines containing 55% and 4 M urea Dense solution: 1% pH 3-5 Ampholines containing 50% and 4 M urea Light solution: 1% pH 3-5 Ampholines containing 10% and 4 M urea Upper electrode solution: 1% phosphoric acid containing 4 M
sucrose sucrose sucrose urea
The LKB model 8101 eleetrofoeusing apparatus is connected to a circulating water b a t h thermostatted at 0°-4 °. The lower electrode solution is pumped into the apparatus. The sample solution is prepared containing approximately 9 mg of protein in a final volume of 5 ml. After the sample solution is loaded into the column, the 10 to 50% linear sucrose gradient is constructed using the light and dense solutions and is pumped into the column. After the loading of the upper electrode solution, the current is applied. Start the eleetrofoeusing with approximately 650 V at 3 mA and gradually increase (over 12 hr) the voltage until 950 V is reached. After approximately 48 hr, the column is fraetionated. As shown in Fig. 3, two peaks of protein and esterase activity are well resolved.
[28]
SEA
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PROTEASES
351
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FIO. 3. Preparative electrofocusing of affinity-purified cortical granule proteases in 4 M urea. O G, IU/ml; • 0, absorbance at 280 nm; A A, pH.
Properties
Stability. The esterase activity in crude cortical granule exudate and purified fractions is stable for weeks at 0 °, pH 4.0 (0.1 M sodium acetate) or for several months (6-8) at pH 4.0, --20 ° (0.05 M sodium acetate-50% glycerol). The prefered method of storage is the latter; the preparation is thus stored as a precipitate in the unfrozen state. Preparations stored frozen as a precipitate at pH 4.0 do not readily redissolve, and considerable material is denatured. The precipitate can simply be harvested from the glycerol by centrifugation and then dissolved in the desired buffer. When crude enzyme preparations are stored at 0 °, pH 8.4, for several days, the specific activity will increase 8- to 10-foldP ,12 Concentrated and purified fractions will exhibit this dramatic increase in specific activity over a time period of minutes to hours depending on the protein concentration. 11 These "activated" preparations (nothing is implied or intended regarding mechanism in the use of this term) have a complete loss of normal biological activity and apparently digest the vitelline layerP Purity and Physical Properties. Solutions of sea urchin egg cortical granule protease purified by isoelectric precipitation contain two proreins, one intensely staining and one faintly staining with Coomassie brilliant blueP These proteins are partially resolved by use of the affinity 12V. D. Vacquier, unpublished observations.
352
P R O T E A S E S FROM GAMETES AND D E V E L O P I N G EMBRYOS
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chromatographic procedure2 Complete separation of the two enzymes has been recently attained with preparative electrofocusing in the presence of 4 M urea. Further biological and biochemical characterization of the resolved proteins is currently in progress. Limited data on the physical properties of the mixture of proteases are relevant to this discussion. The method of Martin and Ames was used to determine the molecular weights of the enzymes in a mixture. A single symmetrical peak of enzymic activity was detected, which corresponded to a molecular weight of 47,000. ~ The presence of only one peak suggested that both vitelline delaminase and sperm receptor hydrolase have the same molecular weight. Unexpectedly, protease preparations displayed a dispersion of molecular weights when chromatographed on Sepharose 4B. The approximate molecular weights ranged between 105 and 10~.s The heterogeneity probably results from aggregation, since rechromatography of the excluded, high-molecular-weight fraction resulted in the reappearance of a fraction of lower molecular weight. The same elution patterns were obtained both at pH 5.6 and 8.0. The observed molecular weight may well be a function of protein concentration. Activators and Inhibitors. Vacquier has reported that the esterase activity of the crude cortical granule exudate prepared by parthenogenetic activation of sea urchin eggs in 0.5 M KC1-0.004 M EGTA pH 8.0 is activated 10-fold when 0.1 M CaC12 is added to divalent free assay medium. The rates with divalent free and 0.1 M MgC12-supplemented assay medium were the same. A 3-fold activation of the divalent free rate was obtained in 0.1 M SrCle. ia Soybean trypsin inhibitor is an effective inhibitor of the crude cortical granule protease activity and of purified fractions. Toluenesulfonyl (tosyl) lysine chloromethyl ketone inhibits the esterase activity of the mixture of enzymes (10 raM, 2 hrs, 4 °, 56% inhibition). Tosyl phenylalanine chloromethyl ketone and phenylmethylsulfonyl fluoride are without effect (0.1 mM and 1.0 raM, respectively, 2 hrs, 4°). With the mixture of proteases, the K~ for p-aminobenzamidine (BAEE as substrate) is 1.6 X 10 -4 M .
The alkaloid nicotine is an effective inhibitor of the esterase activity of the crude cortical granule exudate; protease activity does not seem to be affected. The vitelline delaminase activity is also unaffected, but sperm receptor hydrolase activity is inhibited by nicotine. 14 Thus it 13V. D. Vacquier, Exp. Cell Res. 90, 454 (1975). 24E. J. Carroll, Jr., Amer. Zool. 15, 780 (1975).
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SEA URCHIN EGG PROTEASES
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appears t h a t nicotine is a selective inhibitor of the sperm receptor hydrolase enzyme. These results are significant in t h a t nicotine is a classical polyspermy-including drug, 15 and its mechanism of action is now explained. Additional studies with these inhibitors using the separated proteases is in progress. Specificity. The cortical granule proteases hydrolyze the synthetic esters B A E E and a-N-tosyl arginine methyl ester. Thus, minimally the enzymes hydrolyze N-blocked esters of lysine and arginine. Kinetics. The Km of the crude cortical granule exudate for B A E E is 0.7 mM, and the p H optimum is approximately 8.0. Distribution. The esterase-protease and vitelline delaminase-sperm receptor hydrolase activities have also been found in Lytechinus pictus eggs. 16
Addendum
Fodor et aI. 17 have recently reported the isolation of a protease from Strongylocentrotus purpuratus eortical granule exudate and unfertilized eggs. The protease was solubilized from both starting materials using 2 M K C I - 1 0 % g l y c e r o l - l % butanol-50 m M E D T A - 0 . 2 M Tris at p H 8.0 as an extraction medium. Homogeneous enzyme, similar to bovine trypsin in both susceptibility to inhibitors (diisopropyl phosphofluoridate and soybean trypsin inhibitor) and molecular weight (22,500), was obtained following purification of cortieal granule exudate and unfertilized egg extracts on columns of immobilized soybean trypsin inhibitor. Fodor and co-workers did not perform sperm receptor hydrolase or vitelline delaminase assays; however, as they state, their homogeneous enzyme m a y have been separated from factors which might modulate the functions of the enzymes in fertilization as reported by Carroll and Epel. is Acknowledgments
I thank Professor David Epel for his hospitality and encouragement. The technical assistance of Elizabeth Baker is appreciated. This work was supported by research grants from the National Science Foundation and the Population Council. E. J. Carroll, Jr. is a postdoctoral fellow of the Population Council.
~5F. J. 1,;E. J. ~7E. J. 1~E. J.
Longo and E. Anderson, J. Cell Biol. 46, 308 (1970). Carroll, Jr. and V. D. Vacquier, unpublished observations (1973). Fodor, H. Ako, and K. A. Walsh, Biochemistry 14, 4923 (1975). Carroll, Jr. and D. Epel, Dev. Biol. 44, 22 (1975).