Proc. Natl. Acad. Sci. USA Vol. 74, No. 10, pp. 4214-4218, October 1977
Biochemistry
Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks (protein crosslinking/tyrosine/egg shell/peroxidase/polyspermy)
CHARLES A. FOERDER AND BENNETT M. SHAPIRO Department of Biochemistry, University of Washington, Seattle, Washington 98195
Communicated by Edmond H. Fischer, July 14, 1977
ABSTRACT One feature of fertilization is the alteration of the vitelline layer, by components released from the e, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in thepresence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit peroxidases. A peroxidase, here called the ovoperoxidase, many is released from eggs at fertilization. This enzyme is, inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN3, phenylhydrazine, K4Fe(CN)o, sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein. We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg. Fertilization involves the union of two haploid genomes to make a new diploid individual. To ensure that only one sperm fertilizes an egg, many eggs become modified after penetration by the first sperm. In the case of the sea urchin Strongylocentrotus purpuratus, an immediate block to further sperm entry (polyspermy) is caused by a depolarization of the plasma membrane of the egg (1). After depolarization, the egg surface is rearranged by the cortical reaction (reviewed in refs. 2 and 3), in which secretory vesicles beneath the plasma membrane of the unfertilized egg release their contents in a massive exocytosis. A trypsin-like protease is released to cleave sperm receptors from the egg and thus serve as a second block. to polyspermy (3-5). During the cortical reaction, the vitelline layer that surrounds the egg elevates and is converted into a fertilization membrane. The fertilization membrane becomes altered in its morphology, solubility, and permeability in a discrete, multistep, assembly process that occurs in fixed sequence (6). This elevated structure is another block to sperm entry. The fertilization membrane is formed from the vitelline layer in a reaction with components released from the cortical granules (2, 3, 6). The structure thus produced is resistant to physical deformation (7), solubilization by protein denaturants The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
(3, 8), and proteolytic digestion (9) and is referred to as a hardened fertilization membrane (6). However, the hardened fertilization membrane is dissolved by a hatching enzyme released from the embryo at the blastula stage (10). Thus, the modification of the vitelline layer after fertilization appears to involve a crosslinking reaction that dramatically alters its chemical properties, converting it to a single huge macromolecule. In this paper, we demonstrate that an ovoperoxidase is released from the egg at fertilization to catalyze the formation of di- and trityrosine crosslinks, which harden the fertilization membrane. MATERIALS AND METHODS Procedures for obtaining and handling S. purpuratus gametes were as described (6, 8). Manipulations were at 12°, using seawater passed through a 0.45-,um Millipore filter. Ovoperoxidase Assay. Ovoperoxidase assays were performed in 1 ml containing 18 mM guaiacol, 0.3 mM H202, and 10 mM sodium phosphate at pH 8.0 and 20°. The reaction was started by adding enzyme, and the increase in absorbancy at 436 nm was monitored in a Gilford 420 spectrophotometer (11). All reported values are initial rates; the reaction slows after 15-30 sec. A unit of ovoperoxidase is that required to oxidize 1 'mol of guaiacol per min in a 1-ml assay volume. Crude ovoperoxidase was prepared from a 10-20% suspension of fertilized eggs. Five minutes after fertilization, the eggs were removed by centrifugation at low speed and the supernatant was decanted. Subsequent operations were at 4°. The supernatant was recentrifuged at 17,000 X g for 5 min to remove sperm and residual embryos and then was concentrated 5-fold by ultrafiltration with a PM-10 filter (Amicon). Purification and Analysis of Fertilization Membranes. Fertilization membranes were prepared by a modification of the method of Inoue et al. (12). In a typical purification, 100 ml of a 20% suspension of eggs was centrifuged 10 min after fertilization; the eggs were homogenized in an equal volume of 0.2 mM EDTA/0.2 mM phenylmethylsulfonyl fluoride/5 mM Tris-HCl, pH 8.0 (buffer HB) and centrifuged at 1000 X g for 15 min. The pellet was washed repeatedly in buffer HB until the supernatant was clear and then was resuspended in 20 ml of 6 M guanidine-HCl, heated at 60° for 20 min, and washed again with buffer HB until the supernatant was clear. Phase contrast microscopy of the preparation showed that there was no debris or unlysed eggs. The pellet was then resuspended in 10 ml of buffer HB and applied to the top of a 35-ml discontinuous sucrose gradient, made in buffer HB with 25%, 49%, and 69% sucrose layers. After centrifugation at 52,000 X g for 2 hr at 40, the fertilization membranes were collected at the 49%/69% interface and dialyzed against several changes of 1 Abbreviation: buffer HB, 0.2 mM EDTA/0.2 mM phenylmethylsulfonyl fluoride/5 mM Tris-HCI, pH 8.0. 4214
Biochemistry:
Foerder and Shapiro
Proc. Nati. Acad. Sci. USA 74 (1977)
Table 1. Inhibition of fertilization membrane hardening Inhibitor None Na2SO3 Glycine ethyl ester K4Fe(CN)6 Tyramine N-Acetyl-L-tyrosine O-Methyl-L-tyrosine Phenylhydrazine 3-Amino-1,2,4-triazole KCN NaN3
Concentration
28MAM 36 mM 350 AM 12.5 mM 12.5 mM 12.5 mM 250 nM 27 AM 600 MM 5 mM
Inhibition of hardening, % 0-9 100 99 100 54 99 6 97 92 100 100
A 5% suspension of eggs was mixed with an equivalent amount of 1:100 dilution of sperm in filtered seawater for 10 sec. The substance to be tested for its inhibiting effect on hardening was then added. The mixture was stirred briefly every 2 min for 10 min, after which an equal volume of 100 mM dithiothreitol, pH 8.0, in filtered seawater was added and the eggs were allowed to settle for 10 min at 200. The eggs were mixed at high speed in a Vortex mixer, to remove fertilization membranes that had not hardened, and immediately examined by phase contrast microscopy. The percentage inhibition of hardening was determined by counting 100 eggs and determining the number without fertilization membranes after dithiothreitol treatment. Only batches of eggs that gave at least 90% fertilization in control preparations were used for these assays. a
mM NaN3 at 4°. The fertilization membrane preparation was centrifuged at 12,000 X g for 20 min at 40, and the pellet was lyophilized. This fraction is known as purified hard fertilization membranes. Fertilization membranes were hydrolyzed at 1100 in redistilled 5.7 M HCl for 24 hr in vacuo (after flushing with nitrogen several times) with 2 ml of HCI for each 1 mg of ferTable 2. Release of ovoperoxidase from eggs Peroxidase activity, total units Pellet Supernatant R,*% Experiment 1 15.5 0 0 Eggs 0 0 Sperm 5.8 1.1 16 Eggs + sperm 3.8 1.5 28 Eggs+A23187 Experiment 2 0.1 14.0 0.7 Eggs, trypsin-treated 3.8 6.7 64 Eggs, trypsin-treated + sperm Experiment 1. Five milliliters of a 10% suspension of eggs was mixed with 0.5 ml of a 2% suspension of sperm and incubated for 10 min; 99% of the eggs elevated fertilization membranes. The mixture was centrifuged at 12,000 X g for 10 min (0°) and the supernatant was collected. The pellet was resuspended in 5 ml of filtered seawater and homogenized for 1 min in a Teflon-glass homogenizer. Eggs and sperm were treated separately in a similar fashion. Eggs were also incubated for 10 min in 10MIM A23187 to activate them (28). Experiment 2. Eggs were treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsina at 2.5 ,g/ml to alter the vitelline layer and prevent formation of a fertilization membrane (29). None of the eggs raised a fertilization membrane after fertilization, but all subsequently divided. Supernatant and pellet fractions were isolated as in experiment 1. All preparations were frozen at -20° overnight before assay of ovoperoxidase. * R provides a measure of the percentage of the total peroxidase activity found in the supernatant fraction and is calculated as: 100 X activity in supernatant/(activity in supernatant + activity in pellet).
4215
Table 3. Inhibition of the ovoperoxidase Inhibitor Na2SO3 Glycine ethyl ester KCN 3-Amino-1,2,4-triazole Phenylhydrazine NaN3 K4Fe(CN)6
[I10.5 28 gM 35 mM
20,gM 27,AM 250 nM 5mM 600 AM
Inhibitor concentrations were varied to obtain a value that led to 50% inhibition of ovoperoxidase, lo0.5. Data were obtained with two preparations of crude ovoperoxidase, 30-60 milliunits per assay. Final inhibition was 100% in each case, except with glycine ethyl ester the inhibition was not studied above 0.23 M, which inhibited 75%.
tilization membranes. The acid hydrolysate was evaporated to dryness at 400 in vacuo. RESULTS Inhibition of Fertilization Membrane Hardening. Inhibition of hardening of fertilization membranes renders them susceptible to solubilization in mercaptanes (13, 14) and to breakage by physical forces (15). We developed an assay to assess the effect of various compounds on the hardening process (Table 1). Of eggs tested 10 min after fertilization, 91-100% have intact fertilization membranes by this assay if no inhibitor of hardening is present. Glycine ethyl ester, an inhibitor of certain transglutaminases in itro (16), inhibits hardening of S. purpuratus fertilization membranes (refs. 6 and 14; Table 1). Sodium sulfite and potassium ferrocyanide inhibit fertilization membrane hardening in the sea urchins S. nudus (17) and S. purpuratus (ref. 6; Table 1). Tyramine is also an inhibitor of some transglutaminases (18) as well as of hardening (Table 1), although these inhibitions appear to be by different mechanisms. Transglutaminases require the amine function of tyramine for inhibition, but hardening does not, because N-acetyl-L-tyrosine inhibits hardening (Table 1). On the other hand, the phenolic hydroxyl of tyrosine derivatives is required, because O-methyl-L-tyrosine does not inhibit hardening. Tyrosine could not be tested at similar concentrations, because of its minimal solubility in seawater. Because the tyrosyl derivatives could compete with endogenous protein tyrosines in a crosslinking reaction, and because sulfite and ferrocyanide are classical inhibitors of peroxidases, we suspected that a peroxidase-catalyied reaction, perhaps involving the formation of tyrosine crosslinks, was responsible for hardening. Di- and trityrosine crosslinks have been reported in several systems (19-23). They can be generated by the addition of horseradish peroxidase and H202 to solutions of tyrosine, tyramine, N-acetyl-L-tyrosine, or proteins containing tyrosyl residues (23-25). With this idea in mind, we tested several known inhibitors of peroxidases for their ability to inhibit hardening. Phenylhydrazine proved to be the most potent inhibitor, effective at submicromolar concentrations, whereas potassium cyanide, 3-amino-1,2,4-triazole, and sodium azide inhibited hardening at higher concentrations (Table 1). All of the data are consistent with the possibility that a peroxidase-catalyzed reaction is involved in crosslinking. Ovoperoxidase Release from Fertilized Eggs. This possibility was strengthened by the discovery of a peroxidase associated with egg fractions. The assay utilized guaiacol, a classic substrate for many peroxidases [e.g., myeloperoxidase (26)]. One of the reaction products, 2,2'-dihydroxy-3,3'-dimethoxydiphenyl (27), is similar in structure to dityrosine.
4216
Biochemistry: Foerder and Shapiro
Proc. Natl. Acad. Sci. USA 74 (1977) Table 4. Thin-layer chromatography of fertilization membrane hydrolysis products
Sample
Solvent 1
RF
Solvent 2
Total hydrolysate of hard fertilization membrane 0.15 Dityrosine 0.09 0.03 Trityrosine 0.07 Hydrolysate 0.12 Spot 1 0.07 0.05 0.04 Spot 2 Cellulose phosphate column fractions
RF Dityrosine Trityrosine Peak 1 Peak 2 Peak 3
FIG. 1. Scanning electron micrographs of hardened fertilization membranes. (A) Intact fertilization membrane at 8 min after fertilization. (B) Isolated fertilization membranes. Note the retention of the angular casts after isolation. Microscopy was as described (4). (Bar represents 1 Mm.)
This enzyme, here called the ovoperoxidase, has maximal activity at pH 8, that of the seawater used. The activity is inhibited at hydrogen peroxide concentrations in excess of 300 MM. The distribution of the enzyme in eggs and its release by eliciting the cortical reaction is shown in Table 2. Unfertilized eggs contain all of the activity, which is found in the pellet fraction. There is no activity in sperm. Fertilization leads to release of 16% of the enzyme from the egg fraction, whereas activation of eggs with the divalent ionophore A23187 (6, 28) leads to release of 28% of the enzyme. In neither case is recovery of activity complete after activation, perhaps because of instability of the enzyme; nonetheless, no enzyme is released into the supernatant without the cortical reaction. When eggs are treated with trypsin prior to fertilization, to inhibit elevation of the fertilization membrane (29), 64% of the enzyme is re-
leased at fertilization. These data suggest that the ovoperoxidase is released from eggs at fertilization, perhaps from the cortical granules which undergo exocytosis at that time. The compounds that inhibit hardening in vivo were tested for inhibition of the ovoperoxidase in vitro (Table 3). All inhibitors of hardening tested also inhibited the ovoperoxidase (compare Tables 1 and 3). These results implicate a reaction catalyzed by the ovoperoxidase in fertilization membrane hardening. Tyrosine Crosslinks in the Fertilization Membrane. Several experiments suggest that the ovoperoxidase catalyzes the formation of covalent crosslinks between tyrosyl residues of adjacent polypeptides. Hardened fertilization membranes were isolated by a rigorous procedure (see Materials and Methods) and found to retain a remarkable degree of morphological integrity, as monitored by scanning electron microscopy (Fig. 1).
Acid hydrolysis of fertilization membrane preparations revealed di- and trityrosyl residues, as determined by several
0.29 0.06 0.27 0.27 0.06
For the hydrolysate study, fertilization membrane hydrolysate (1 mg) was resuspended in NH40H/ethanol/water, 2:6:17 (vol/vol), and transferred to a 20 X 20 cm cellulose thin-layer plate (Kontes). The solvent for the first dimension was isopropanol/NH40H/water, 8:1:1 (vol/vol). For the second dimension it was 1-butanol/acetic acid/H20, 4:1:1 (vol/vol). A broad zone of fluorescence was seen in the initial chromatogram; it was scraped off, extracted twice with 25 M1 of NH40H/EtOH/H20, 2:16:17 (vol/vol), and rechromatographed as before. The plate was exposed to NH3 vapor and examined with an ultraviolet light (240-380 nm) to detect di- and trityrosine which were prepared (24) and run on the same chromatographic plate for comparison. For the column study, fractions from the cellulose phosphate column (Fig. 2) were pooled and dried at 400 in vacuo. The residue was resuspended in 0.1 M HC1, and salt was removed in a 1.6 X 9.0 cm column of Dowex AG 50W-X8 (19). Desalted fractions were dried under a stream of nitrogen at 400, resuspended in 0.1 ml of distilled water, applied to a silica gel thin-layer plate (Kontes), and compared with the standard di- and trityrosine mixture after development in 1-butanol/acetic acid/H20, 4:1:1 (vol/vol). Fluorescent spots were detected as above.
criteria. Two components were found to travel, in a two-dimensional thin-layer chromatography system, similar to authentic di- and trityrosine (Table 4). Both fluoresced with a blue color when illuminated with ultraviolet light at alkaline pH (19), but not at acid pH. Dityrosine migrated with an RF value close to that of spot 1 of the fertilization membrane hydrolysate, and trityrosine migrated close to spot 2. The slight difference in RF
between the sample and the standards probably reflects the presence of other amino acids in the hydrolysate of hard fertilization membranes. In fact, after one two-dimensional chromatogram, there was only a broad zone of fluorescence, which, upon subsequent chromatography, clearly separated the two components, as shown in Table 4. By cellulose phosphate chromatography, compounds with the characteristic fluorescence properties migrated in the position of authentic di- and trityrosine (Fig. 2). The fractions isolated from this column, when pooled, ran in a one-dimensional thin-layer chromatography system much like authentic di- and trityrosine (Table 4). The reason for dityrosine in the hydrolysate being found in two peaks (1 and 2) is not known (Fig. 2 and Table 4). We conclude that hardened fertilization membranes do, indeed, contain di- and trityrosine. The amount of dityrosine present in hardened fertilization membrane preparations was estimated by conventional amino acid analysis with authentic dityrosine as standard. Means of two analyses are shown in Table 5; the data from the two runs, with the exception of methionine and cysteine, were nearly
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Foerder and Shapiro
4217
Table 5. Amino acid composition of hardened fertilization membrane
0) a)
0 L-
10
20
30
40
50
60
70
Fraction
FIG. 2. Cellulose phosphate chromatography of amino acid hydrolysates of hard fertilization membranes. To 10 mg of hydrolysate, before evaporation, 2 Ml of [3Hltyrosine (New England Nuclear) was added. The dried residue was resuspended in 0.2 M acetic acid to a final conductivity of less than 5 mS. Cellulose phosphate (Cellex-P, Bio-Rad) chromatography was performed on a 0.5 X 55 cm column, as described (19). After the column was washed in 150 ml of 0.2 M acetic acid, a 400-ml linear gradient of 0-0.4 M NaCl in 0.2 M acetic acid was run at a rate of 20 ml/hr at 200. Fractions (5 ml) were monitored for conductivity, absorbance at 280 nm, and radioactivity. Radioactivity was estimated in 50-Ml aliquots, in a Beckman LS-230 scintillation counter, in 10 ml of Triton-Omnifluor scintillant. The fractions also were monitored for fluorescence in an Aminco fluorocolorimeter after addition of 0.5 ml of concentrated NH40H to increase the pH to 9-10. Fluorescence was excited at 320-390 nm and measured at 410-480 nm (19). Tyrosine was identified by the mobility of the [3H]tyrosine standard. Authentic dityrosine migrated at peak 1, trityrosine at peak 3.
identical. The amount of dityrosine was estimated by the area under a small doublet peak that comigrated with authentic dityrosine. On the basis of these data there appeared to be one dityrosine crosslink per 55,000 daltons of fertilization membrane protein. However, because the dityrosine peak was small and broad, with a double maximum, the value is tentative. DISCUSSION The data presented are consistent with the model depicted in Fig. 3. Ovoperoxidase is released from eggs after fertilization, presumably from the cortical granules. Inhibition studies clearly demonstrate that inhibitors of hardening inhibit the ovoperoxidase. Classical hardening inhibitors such as glycine ethyl ester (6, 14), sodium sulfite, and potassium ferrocyanide (17) are inhibitors of the ovoperoxidase in vitro (Table 1). Additionally, the inhibition data indicate that the phenolic hydroxyl group of tyrosine is required to prevent hardening. This is consistent with the proposed mechanism of peroxidase-catalyzed dityrosine formation (24). Finally, we have demonstrated the presence of di- and trityrosyl residues in the hardened fertilization membranes. About 15% of the tyrosyl residues of hardened fertilization membranes are in dityrosine linkages, with one crosslink occurring every 27,500 daltons of polypeptide chain, on the average. Unequivocal evidence for the role of dityrosine as the functional crosslink depends upon its absence in fertilization membranes before hardening. Di- and trityrosyl residues are widely distributed in proteins, including resilin, the elastic protein in many arthropod ligaments (19), in silk fibroin and keratin of tussah silk moths (20), elastin (30) and another connective tissue protein from mammals (21), and in the adhesive discs of the sea mussel (31). Dityrosine has also been found in egg envelopes of dragonflies (22) and Drosophila melanogaster (32). A "peroxidatic activity of catalase" was found (33) in the cortical granules of unfertilized eggs of the sea urchins Temnopleurus toreumatcus and
Amino acid Asx Thr Ser Glx Pro Gly Ala Cys* Val Met* Ile Leu
Tyr
Residues, no. 71.7 23.9 31.7 53.0 45.2 55.2 21.3 11.3 30.0 1.7 18.7 14.8 11.3 12.2 4.3 8.7 21.3 1.0
Phe His Lys Arg DiTyr Minimum molecular weight 54,350 Amino acid- analysis was performed on acid hydrolysates of two different hard fertilization membrane preparations. A standard diand trityrosine mixture (24) was fractionated by cellulose phosphate chromatography. Concentrations were determined by their known absorbancy indices (19), to obtain standard values for the ninhydrin reaction. Samples were run on a Durrum D500 analyzer with a DC4A resin. Values are the means for the two preparations and are normalized to one dityrosine residue. We are grateful to Lowell Ericsson and Richard Granberg for the amino acid analysis. * With the exceptions of cysteine and methionine (no special precautions were taken to protect these residues), there was excellent agreement between the analyses.
Hemicentrotus pulcherrimus. Oocytes of the brine shrimp Artemia salina release a peroxidase into the perivitelline space that is apparently responsible for hardening the egg envelope (34). Inhibition of this peroxidase results in egg envelopes with less mechanical resistance and higher permeability than untreated envelopes. The idea that hardening of the fertilization membrane might be an oxidative process was proposed over 20 years ago by Motomura (17). A question raised by our studies concerns the source of the H202 for the ovoperoxidase. Although sperm of some species generate H202 under certain conditions (35), parthenogenetic activation of the egg with the divalent ionophore A23187 leads to a hardened fertilization membrane (6), suggesting that sperm do not play an essential role. An interesting candidate for production of H202 is glucose oxidase. An enzyme that can produce free glucose from ,B-1,3 glucose polymers is released from S. purpuratus eggs upon fertilization (36). Glucose produced by this enzyme could then be used for production of H202 by a glucose oxidase. Such a linked oxidation scheme is utilized by
FIG. 3. Schematic of the crosslinking in fertilization membranes. The average amount of protein per dityrosine crosslink is shown as a total of 55,000 daltons, distributed equally between two crosslinked polypeptides; of course, many possible arrangements may yield the same average value.
4218
Biochemistry: Foerder and Shapiro
mold, Caldariomyces fumago, in a chlorination reaction (37). Smith and Klebanoff (38) have demonstrated that peroxidase activity in rat uterine fluid is spermicidal for mammalian sperm. They also showed that this activity could be replaced by myeloperoxidase and lactoperoxidase. B. M. Shapiro (unpublished data) has found lactoperoxidase to be spermicidal for S. purpuratus in the presence of H202 and iodide ions. It is interesting to speculate, therefore, that ovoperoxidase may perform a second function-acting as a spermicidal agent and thus as an additional block to polyspermy. This function may extend to other eggs containing cortical granules, such as mammalian eggs.
a
Note Added in Proof. We recently became aware that Glenn Hall, Department of Chemistry, University of California, Berkeley, has obtained similar data on the hardening mechanism (G. Hall, personal
communication). We are grateful to William Parson, Seymour Klebanoff, E. M. Eddy, and Christopher Gabel for suggestions and for careful review and criticism of the manuscript. We are grateful to Beverly Foerder for assistance with the scanning electron microscopy. This research was supported in part by National Science Foundation Grant BMS 01463 and National Institutes of Health Biochemical Training Grant 5 T01 GM 00052 to the Department of Biochemistry. A portion of this work was presented at the Annual Meeting of the American Society of Biological Chemists in April 1977 [Fed. Proc. 36,926 (1977)]. 1. Jaffe, L. A. (1976) Nature 261, 68-71. 2. Giudice, G. (1973) Developmental Biology of the Sea Urchin Embryo (Academic Press, New York). 3. Runnstrom, J. (1966) Adv. Morphog. 5,221-325. 4. Hagstrom, B. E. (1956) Ark. Zool. 10, 307-315. 5. Vacquier, V. D., Tegner, M. J. & Epel, D. (1972) Nature 240, 352-353. 6. Veron, M., Foerder, C., Eddy, E. M. & Shapiro, B. M. (1977) Cell 10,321-328. 7. Markman, B. (1958) Acta Zool. (Stockholm) 39, 103-115. 8. Shapiro, B. M. (1975) Dev. Biol. 46,88-102. 9. Runnstr6m, J. L., Monne, L. & Broman, L. (1944) Ark. Zool. 35A, No. 3, 1-32. 10. Ishida, J. (1936) Annot. Zool. Jpn. 15,453-459. 11. Bergmeyer, H. U., Gawehn, K. & Grossl, M. (1974) in Methods of Biochemical Analysis, ed. Bergmeyer, H. U. (Academic Press, New York), pp. 494-495.
Froc.. Natl. Acad. Sa. USA 74 (1977) 12. Inoue, S., Hardy, J. P., Cousineau, G. H. & Bal, A. K. (1976) Exp. Cell Res. 48, 248-251. 13. Lallier, R. (1970) Exp. Cell Res. 63,460-462. 14. Lallier, R. (1971) Experientia 27, 1323-1324. 15. Motomura, I. (1954) Sci. Rep. Tohoku Univ. Ser. 4 20, 219225. 16. Doolittle, R. F. (1973) Adv. Protein Chem. 27, 1-109. 17. Motomura, I. (1954) Sci. Rep. Tohoku Univ. Ser. 4 20, 219225. 18. Dutton, A. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. USA 72, 2568-2571. 19. Andersen, S. 0. (1966) Acta Physiol. Scand. Suppl. 263 66, 1-81. 20. Raven, D. J., Earland, C. & Little, M. (1971) Biochim. Biophys. Acta 251, 96-99. 21. Keeley, F. W. & LaBella, F. S. (1972) Biochim. Biophys. Acta 263,52-59. 22. Kawasaki, H., Sato, H. & Suzuki, M. (1974) Insect Biochem. 4, 99-111. 23. Aeschbach, R., Amado, R. & Neukom, H. (1976) Biochim. Biophys. Acta 439,292-301. 24. Gross, A. J. & Sizer, I. W. (1959) J. Biol. Chem. 234, 16111614. 25. LaBella, F., Waykole, P. & Queen, G. (1968) Biochem. Biophys. Res. Commun. 30,333-338. 26. Himmelhoch, S. R., Evans, W. H., Mage, M. G. & Peterson, E. A. (1969) Biochemistry 8,914-921. 27. Saunders, B. C., Holmes-Siedle, A. G. & Stark, B. P. (1964) Peroxidase-The Properties and Uses of a Versatile Enzyme and of Some Related Catalysts (Butterworths, Washington, DC). 28. Steinhardt, R. A., Epel, D., Carroll, E. J. & Yanagimachi, R. (1974) Nature 252,41-48. 29. Epel, D. (1970) Exp. Cell Res. 61, 69-70. 30. LaBella, F., Keeley, F., Vivian, S. & Thornhill, D. (1967) Biochem. Biophys. Res. Commun. 26,748-753. 31. DeVore, D. P. & Gruebel, R. J. (1977) Fed Proc. 36,679. 32. Petri, W. H., Wyman, A. R. & Kafatos, F. C. (1976) Dev. Biol. 49, 185-199. 33. Katsura, S. & Tominaga, A. (1974) Dev. Biol. 40, 292-297. 34. Roels, F. (1971) Exp. Cell Res. 69,452-456. 35. Klebanoff, S. J. & Smith, D. C. (1970) Biol. Reprod. 3, 236242. 36. Epel, D., Weaver, A. M., Muchmore, A. V. & Schimke, R. T. (1969) Science 163, 294-296. 37. Shaw, P. D. & Hager, L. P. (1961) J. Biol. Chem. 236, 16261630. 38. Smith, D. C. & Klebanoff, S. J. (1970) Biol. Reprod. 3, 229235.
Biochemistry
Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks (protein crosslinking/tyrosine/egg shell/peroxidase/polyspermy)
CHARLES A. FOERDER AND BENNETT M. SHAPIRO Department of Biochemistry, University of Washington, Seattle, Washington 98195
Communicated by Edmond H. Fischer, July 14, 1977
ABSTRACT One feature of fertilization is the alteration of the vitelline layer, by components released from the e, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in thepresence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit peroxidases. A peroxidase, here called the ovoperoxidase, many is released from eggs at fertilization. This enzyme is, inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN3, phenylhydrazine, K4Fe(CN)o, sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein. We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg. Fertilization involves the union of two haploid genomes to make a new diploid individual. To ensure that only one sperm fertilizes an egg, many eggs become modified after penetration by the first sperm. In the case of the sea urchin Strongylocentrotus purpuratus, an immediate block to further sperm entry (polyspermy) is caused by a depolarization of the plasma membrane of the egg (1). After depolarization, the egg surface is rearranged by the cortical reaction (reviewed in refs. 2 and 3), in which secretory vesicles beneath the plasma membrane of the unfertilized egg release their contents in a massive exocytosis. A trypsin-like protease is released to cleave sperm receptors from the egg and thus serve as a second block. to polyspermy (3-5). During the cortical reaction, the vitelline layer that surrounds the egg elevates and is converted into a fertilization membrane. The fertilization membrane becomes altered in its morphology, solubility, and permeability in a discrete, multistep, assembly process that occurs in fixed sequence (6). This elevated structure is another block to sperm entry. The fertilization membrane is formed from the vitelline layer in a reaction with components released from the cortical granules (2, 3, 6). The structure thus produced is resistant to physical deformation (7), solubilization by protein denaturants The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
(3, 8), and proteolytic digestion (9) and is referred to as a hardened fertilization membrane (6). However, the hardened fertilization membrane is dissolved by a hatching enzyme released from the embryo at the blastula stage (10). Thus, the modification of the vitelline layer after fertilization appears to involve a crosslinking reaction that dramatically alters its chemical properties, converting it to a single huge macromolecule. In this paper, we demonstrate that an ovoperoxidase is released from the egg at fertilization to catalyze the formation of di- and trityrosine crosslinks, which harden the fertilization membrane. MATERIALS AND METHODS Procedures for obtaining and handling S. purpuratus gametes were as described (6, 8). Manipulations were at 12°, using seawater passed through a 0.45-,um Millipore filter. Ovoperoxidase Assay. Ovoperoxidase assays were performed in 1 ml containing 18 mM guaiacol, 0.3 mM H202, and 10 mM sodium phosphate at pH 8.0 and 20°. The reaction was started by adding enzyme, and the increase in absorbancy at 436 nm was monitored in a Gilford 420 spectrophotometer (11). All reported values are initial rates; the reaction slows after 15-30 sec. A unit of ovoperoxidase is that required to oxidize 1 'mol of guaiacol per min in a 1-ml assay volume. Crude ovoperoxidase was prepared from a 10-20% suspension of fertilized eggs. Five minutes after fertilization, the eggs were removed by centrifugation at low speed and the supernatant was decanted. Subsequent operations were at 4°. The supernatant was recentrifuged at 17,000 X g for 5 min to remove sperm and residual embryos and then was concentrated 5-fold by ultrafiltration with a PM-10 filter (Amicon). Purification and Analysis of Fertilization Membranes. Fertilization membranes were prepared by a modification of the method of Inoue et al. (12). In a typical purification, 100 ml of a 20% suspension of eggs was centrifuged 10 min after fertilization; the eggs were homogenized in an equal volume of 0.2 mM EDTA/0.2 mM phenylmethylsulfonyl fluoride/5 mM Tris-HCl, pH 8.0 (buffer HB) and centrifuged at 1000 X g for 15 min. The pellet was washed repeatedly in buffer HB until the supernatant was clear and then was resuspended in 20 ml of 6 M guanidine-HCl, heated at 60° for 20 min, and washed again with buffer HB until the supernatant was clear. Phase contrast microscopy of the preparation showed that there was no debris or unlysed eggs. The pellet was then resuspended in 10 ml of buffer HB and applied to the top of a 35-ml discontinuous sucrose gradient, made in buffer HB with 25%, 49%, and 69% sucrose layers. After centrifugation at 52,000 X g for 2 hr at 40, the fertilization membranes were collected at the 49%/69% interface and dialyzed against several changes of 1 Abbreviation: buffer HB, 0.2 mM EDTA/0.2 mM phenylmethylsulfonyl fluoride/5 mM Tris-HCI, pH 8.0. 4214
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Proc. Nati. Acad. Sci. USA 74 (1977)
Table 1. Inhibition of fertilization membrane hardening Inhibitor None Na2SO3 Glycine ethyl ester K4Fe(CN)6 Tyramine N-Acetyl-L-tyrosine O-Methyl-L-tyrosine Phenylhydrazine 3-Amino-1,2,4-triazole KCN NaN3
Concentration
28MAM 36 mM 350 AM 12.5 mM 12.5 mM 12.5 mM 250 nM 27 AM 600 MM 5 mM
Inhibition of hardening, % 0-9 100 99 100 54 99 6 97 92 100 100
A 5% suspension of eggs was mixed with an equivalent amount of 1:100 dilution of sperm in filtered seawater for 10 sec. The substance to be tested for its inhibiting effect on hardening was then added. The mixture was stirred briefly every 2 min for 10 min, after which an equal volume of 100 mM dithiothreitol, pH 8.0, in filtered seawater was added and the eggs were allowed to settle for 10 min at 200. The eggs were mixed at high speed in a Vortex mixer, to remove fertilization membranes that had not hardened, and immediately examined by phase contrast microscopy. The percentage inhibition of hardening was determined by counting 100 eggs and determining the number without fertilization membranes after dithiothreitol treatment. Only batches of eggs that gave at least 90% fertilization in control preparations were used for these assays. a
mM NaN3 at 4°. The fertilization membrane preparation was centrifuged at 12,000 X g for 20 min at 40, and the pellet was lyophilized. This fraction is known as purified hard fertilization membranes. Fertilization membranes were hydrolyzed at 1100 in redistilled 5.7 M HCl for 24 hr in vacuo (after flushing with nitrogen several times) with 2 ml of HCI for each 1 mg of ferTable 2. Release of ovoperoxidase from eggs Peroxidase activity, total units Pellet Supernatant R,*% Experiment 1 15.5 0 0 Eggs 0 0 Sperm 5.8 1.1 16 Eggs + sperm 3.8 1.5 28 Eggs+A23187 Experiment 2 0.1 14.0 0.7 Eggs, trypsin-treated 3.8 6.7 64 Eggs, trypsin-treated + sperm Experiment 1. Five milliliters of a 10% suspension of eggs was mixed with 0.5 ml of a 2% suspension of sperm and incubated for 10 min; 99% of the eggs elevated fertilization membranes. The mixture was centrifuged at 12,000 X g for 10 min (0°) and the supernatant was collected. The pellet was resuspended in 5 ml of filtered seawater and homogenized for 1 min in a Teflon-glass homogenizer. Eggs and sperm were treated separately in a similar fashion. Eggs were also incubated for 10 min in 10MIM A23187 to activate them (28). Experiment 2. Eggs were treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsina at 2.5 ,g/ml to alter the vitelline layer and prevent formation of a fertilization membrane (29). None of the eggs raised a fertilization membrane after fertilization, but all subsequently divided. Supernatant and pellet fractions were isolated as in experiment 1. All preparations were frozen at -20° overnight before assay of ovoperoxidase. * R provides a measure of the percentage of the total peroxidase activity found in the supernatant fraction and is calculated as: 100 X activity in supernatant/(activity in supernatant + activity in pellet).
4215
Table 3. Inhibition of the ovoperoxidase Inhibitor Na2SO3 Glycine ethyl ester KCN 3-Amino-1,2,4-triazole Phenylhydrazine NaN3 K4Fe(CN)6
[I10.5 28 gM 35 mM
20,gM 27,AM 250 nM 5mM 600 AM
Inhibitor concentrations were varied to obtain a value that led to 50% inhibition of ovoperoxidase, lo0.5. Data were obtained with two preparations of crude ovoperoxidase, 30-60 milliunits per assay. Final inhibition was 100% in each case, except with glycine ethyl ester the inhibition was not studied above 0.23 M, which inhibited 75%.
tilization membranes. The acid hydrolysate was evaporated to dryness at 400 in vacuo. RESULTS Inhibition of Fertilization Membrane Hardening. Inhibition of hardening of fertilization membranes renders them susceptible to solubilization in mercaptanes (13, 14) and to breakage by physical forces (15). We developed an assay to assess the effect of various compounds on the hardening process (Table 1). Of eggs tested 10 min after fertilization, 91-100% have intact fertilization membranes by this assay if no inhibitor of hardening is present. Glycine ethyl ester, an inhibitor of certain transglutaminases in itro (16), inhibits hardening of S. purpuratus fertilization membranes (refs. 6 and 14; Table 1). Sodium sulfite and potassium ferrocyanide inhibit fertilization membrane hardening in the sea urchins S. nudus (17) and S. purpuratus (ref. 6; Table 1). Tyramine is also an inhibitor of some transglutaminases (18) as well as of hardening (Table 1), although these inhibitions appear to be by different mechanisms. Transglutaminases require the amine function of tyramine for inhibition, but hardening does not, because N-acetyl-L-tyrosine inhibits hardening (Table 1). On the other hand, the phenolic hydroxyl of tyrosine derivatives is required, because O-methyl-L-tyrosine does not inhibit hardening. Tyrosine could not be tested at similar concentrations, because of its minimal solubility in seawater. Because the tyrosyl derivatives could compete with endogenous protein tyrosines in a crosslinking reaction, and because sulfite and ferrocyanide are classical inhibitors of peroxidases, we suspected that a peroxidase-catalyied reaction, perhaps involving the formation of tyrosine crosslinks, was responsible for hardening. Di- and trityrosine crosslinks have been reported in several systems (19-23). They can be generated by the addition of horseradish peroxidase and H202 to solutions of tyrosine, tyramine, N-acetyl-L-tyrosine, or proteins containing tyrosyl residues (23-25). With this idea in mind, we tested several known inhibitors of peroxidases for their ability to inhibit hardening. Phenylhydrazine proved to be the most potent inhibitor, effective at submicromolar concentrations, whereas potassium cyanide, 3-amino-1,2,4-triazole, and sodium azide inhibited hardening at higher concentrations (Table 1). All of the data are consistent with the possibility that a peroxidase-catalyzed reaction is involved in crosslinking. Ovoperoxidase Release from Fertilized Eggs. This possibility was strengthened by the discovery of a peroxidase associated with egg fractions. The assay utilized guaiacol, a classic substrate for many peroxidases [e.g., myeloperoxidase (26)]. One of the reaction products, 2,2'-dihydroxy-3,3'-dimethoxydiphenyl (27), is similar in structure to dityrosine.
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Proc. Natl. Acad. Sci. USA 74 (1977) Table 4. Thin-layer chromatography of fertilization membrane hydrolysis products
Sample
Solvent 1
RF
Solvent 2
Total hydrolysate of hard fertilization membrane 0.15 Dityrosine 0.09 0.03 Trityrosine 0.07 Hydrolysate 0.12 Spot 1 0.07 0.05 0.04 Spot 2 Cellulose phosphate column fractions
RF Dityrosine Trityrosine Peak 1 Peak 2 Peak 3
FIG. 1. Scanning electron micrographs of hardened fertilization membranes. (A) Intact fertilization membrane at 8 min after fertilization. (B) Isolated fertilization membranes. Note the retention of the angular casts after isolation. Microscopy was as described (4). (Bar represents 1 Mm.)
This enzyme, here called the ovoperoxidase, has maximal activity at pH 8, that of the seawater used. The activity is inhibited at hydrogen peroxide concentrations in excess of 300 MM. The distribution of the enzyme in eggs and its release by eliciting the cortical reaction is shown in Table 2. Unfertilized eggs contain all of the activity, which is found in the pellet fraction. There is no activity in sperm. Fertilization leads to release of 16% of the enzyme from the egg fraction, whereas activation of eggs with the divalent ionophore A23187 (6, 28) leads to release of 28% of the enzyme. In neither case is recovery of activity complete after activation, perhaps because of instability of the enzyme; nonetheless, no enzyme is released into the supernatant without the cortical reaction. When eggs are treated with trypsin prior to fertilization, to inhibit elevation of the fertilization membrane (29), 64% of the enzyme is re-
leased at fertilization. These data suggest that the ovoperoxidase is released from eggs at fertilization, perhaps from the cortical granules which undergo exocytosis at that time. The compounds that inhibit hardening in vivo were tested for inhibition of the ovoperoxidase in vitro (Table 3). All inhibitors of hardening tested also inhibited the ovoperoxidase (compare Tables 1 and 3). These results implicate a reaction catalyzed by the ovoperoxidase in fertilization membrane hardening. Tyrosine Crosslinks in the Fertilization Membrane. Several experiments suggest that the ovoperoxidase catalyzes the formation of covalent crosslinks between tyrosyl residues of adjacent polypeptides. Hardened fertilization membranes were isolated by a rigorous procedure (see Materials and Methods) and found to retain a remarkable degree of morphological integrity, as monitored by scanning electron microscopy (Fig. 1).
Acid hydrolysis of fertilization membrane preparations revealed di- and trityrosyl residues, as determined by several
0.29 0.06 0.27 0.27 0.06
For the hydrolysate study, fertilization membrane hydrolysate (1 mg) was resuspended in NH40H/ethanol/water, 2:6:17 (vol/vol), and transferred to a 20 X 20 cm cellulose thin-layer plate (Kontes). The solvent for the first dimension was isopropanol/NH40H/water, 8:1:1 (vol/vol). For the second dimension it was 1-butanol/acetic acid/H20, 4:1:1 (vol/vol). A broad zone of fluorescence was seen in the initial chromatogram; it was scraped off, extracted twice with 25 M1 of NH40H/EtOH/H20, 2:16:17 (vol/vol), and rechromatographed as before. The plate was exposed to NH3 vapor and examined with an ultraviolet light (240-380 nm) to detect di- and trityrosine which were prepared (24) and run on the same chromatographic plate for comparison. For the column study, fractions from the cellulose phosphate column (Fig. 2) were pooled and dried at 400 in vacuo. The residue was resuspended in 0.1 M HC1, and salt was removed in a 1.6 X 9.0 cm column of Dowex AG 50W-X8 (19). Desalted fractions were dried under a stream of nitrogen at 400, resuspended in 0.1 ml of distilled water, applied to a silica gel thin-layer plate (Kontes), and compared with the standard di- and trityrosine mixture after development in 1-butanol/acetic acid/H20, 4:1:1 (vol/vol). Fluorescent spots were detected as above.
criteria. Two components were found to travel, in a two-dimensional thin-layer chromatography system, similar to authentic di- and trityrosine (Table 4). Both fluoresced with a blue color when illuminated with ultraviolet light at alkaline pH (19), but not at acid pH. Dityrosine migrated with an RF value close to that of spot 1 of the fertilization membrane hydrolysate, and trityrosine migrated close to spot 2. The slight difference in RF
between the sample and the standards probably reflects the presence of other amino acids in the hydrolysate of hard fertilization membranes. In fact, after one two-dimensional chromatogram, there was only a broad zone of fluorescence, which, upon subsequent chromatography, clearly separated the two components, as shown in Table 4. By cellulose phosphate chromatography, compounds with the characteristic fluorescence properties migrated in the position of authentic di- and trityrosine (Fig. 2). The fractions isolated from this column, when pooled, ran in a one-dimensional thin-layer chromatography system much like authentic di- and trityrosine (Table 4). The reason for dityrosine in the hydrolysate being found in two peaks (1 and 2) is not known (Fig. 2 and Table 4). We conclude that hardened fertilization membranes do, indeed, contain di- and trityrosine. The amount of dityrosine present in hardened fertilization membrane preparations was estimated by conventional amino acid analysis with authentic dityrosine as standard. Means of two analyses are shown in Table 5; the data from the two runs, with the exception of methionine and cysteine, were nearly
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Foerder and Shapiro
4217
Table 5. Amino acid composition of hardened fertilization membrane
0) a)
0 L-
10
20
30
40
50
60
70
Fraction
FIG. 2. Cellulose phosphate chromatography of amino acid hydrolysates of hard fertilization membranes. To 10 mg of hydrolysate, before evaporation, 2 Ml of [3Hltyrosine (New England Nuclear) was added. The dried residue was resuspended in 0.2 M acetic acid to a final conductivity of less than 5 mS. Cellulose phosphate (Cellex-P, Bio-Rad) chromatography was performed on a 0.5 X 55 cm column, as described (19). After the column was washed in 150 ml of 0.2 M acetic acid, a 400-ml linear gradient of 0-0.4 M NaCl in 0.2 M acetic acid was run at a rate of 20 ml/hr at 200. Fractions (5 ml) were monitored for conductivity, absorbance at 280 nm, and radioactivity. Radioactivity was estimated in 50-Ml aliquots, in a Beckman LS-230 scintillation counter, in 10 ml of Triton-Omnifluor scintillant. The fractions also were monitored for fluorescence in an Aminco fluorocolorimeter after addition of 0.5 ml of concentrated NH40H to increase the pH to 9-10. Fluorescence was excited at 320-390 nm and measured at 410-480 nm (19). Tyrosine was identified by the mobility of the [3H]tyrosine standard. Authentic dityrosine migrated at peak 1, trityrosine at peak 3.
identical. The amount of dityrosine was estimated by the area under a small doublet peak that comigrated with authentic dityrosine. On the basis of these data there appeared to be one dityrosine crosslink per 55,000 daltons of fertilization membrane protein. However, because the dityrosine peak was small and broad, with a double maximum, the value is tentative. DISCUSSION The data presented are consistent with the model depicted in Fig. 3. Ovoperoxidase is released from eggs after fertilization, presumably from the cortical granules. Inhibition studies clearly demonstrate that inhibitors of hardening inhibit the ovoperoxidase. Classical hardening inhibitors such as glycine ethyl ester (6, 14), sodium sulfite, and potassium ferrocyanide (17) are inhibitors of the ovoperoxidase in vitro (Table 1). Additionally, the inhibition data indicate that the phenolic hydroxyl group of tyrosine is required to prevent hardening. This is consistent with the proposed mechanism of peroxidase-catalyzed dityrosine formation (24). Finally, we have demonstrated the presence of di- and trityrosyl residues in the hardened fertilization membranes. About 15% of the tyrosyl residues of hardened fertilization membranes are in dityrosine linkages, with one crosslink occurring every 27,500 daltons of polypeptide chain, on the average. Unequivocal evidence for the role of dityrosine as the functional crosslink depends upon its absence in fertilization membranes before hardening. Di- and trityrosyl residues are widely distributed in proteins, including resilin, the elastic protein in many arthropod ligaments (19), in silk fibroin and keratin of tussah silk moths (20), elastin (30) and another connective tissue protein from mammals (21), and in the adhesive discs of the sea mussel (31). Dityrosine has also been found in egg envelopes of dragonflies (22) and Drosophila melanogaster (32). A "peroxidatic activity of catalase" was found (33) in the cortical granules of unfertilized eggs of the sea urchins Temnopleurus toreumatcus and
Amino acid Asx Thr Ser Glx Pro Gly Ala Cys* Val Met* Ile Leu
Tyr
Residues, no. 71.7 23.9 31.7 53.0 45.2 55.2 21.3 11.3 30.0 1.7 18.7 14.8 11.3 12.2 4.3 8.7 21.3 1.0
Phe His Lys Arg DiTyr Minimum molecular weight 54,350 Amino acid- analysis was performed on acid hydrolysates of two different hard fertilization membrane preparations. A standard diand trityrosine mixture (24) was fractionated by cellulose phosphate chromatography. Concentrations were determined by their known absorbancy indices (19), to obtain standard values for the ninhydrin reaction. Samples were run on a Durrum D500 analyzer with a DC4A resin. Values are the means for the two preparations and are normalized to one dityrosine residue. We are grateful to Lowell Ericsson and Richard Granberg for the amino acid analysis. * With the exceptions of cysteine and methionine (no special precautions were taken to protect these residues), there was excellent agreement between the analyses.
Hemicentrotus pulcherrimus. Oocytes of the brine shrimp Artemia salina release a peroxidase into the perivitelline space that is apparently responsible for hardening the egg envelope (34). Inhibition of this peroxidase results in egg envelopes with less mechanical resistance and higher permeability than untreated envelopes. The idea that hardening of the fertilization membrane might be an oxidative process was proposed over 20 years ago by Motomura (17). A question raised by our studies concerns the source of the H202 for the ovoperoxidase. Although sperm of some species generate H202 under certain conditions (35), parthenogenetic activation of the egg with the divalent ionophore A23187 leads to a hardened fertilization membrane (6), suggesting that sperm do not play an essential role. An interesting candidate for production of H202 is glucose oxidase. An enzyme that can produce free glucose from ,B-1,3 glucose polymers is released from S. purpuratus eggs upon fertilization (36). Glucose produced by this enzyme could then be used for production of H202 by a glucose oxidase. Such a linked oxidation scheme is utilized by
FIG. 3. Schematic of the crosslinking in fertilization membranes. The average amount of protein per dityrosine crosslink is shown as a total of 55,000 daltons, distributed equally between two crosslinked polypeptides; of course, many possible arrangements may yield the same average value.
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Biochemistry: Foerder and Shapiro
mold, Caldariomyces fumago, in a chlorination reaction (37). Smith and Klebanoff (38) have demonstrated that peroxidase activity in rat uterine fluid is spermicidal for mammalian sperm. They also showed that this activity could be replaced by myeloperoxidase and lactoperoxidase. B. M. Shapiro (unpublished data) has found lactoperoxidase to be spermicidal for S. purpuratus in the presence of H202 and iodide ions. It is interesting to speculate, therefore, that ovoperoxidase may perform a second function-acting as a spermicidal agent and thus as an additional block to polyspermy. This function may extend to other eggs containing cortical granules, such as mammalian eggs.
a
Note Added in Proof. We recently became aware that Glenn Hall, Department of Chemistry, University of California, Berkeley, has obtained similar data on the hardening mechanism (G. Hall, personal
communication). We are grateful to William Parson, Seymour Klebanoff, E. M. Eddy, and Christopher Gabel for suggestions and for careful review and criticism of the manuscript. We are grateful to Beverly Foerder for assistance with the scanning electron microscopy. This research was supported in part by National Science Foundation Grant BMS 01463 and National Institutes of Health Biochemical Training Grant 5 T01 GM 00052 to the Department of Biochemistry. A portion of this work was presented at the Annual Meeting of the American Society of Biological Chemists in April 1977 [Fed. Proc. 36,926 (1977)]. 1. Jaffe, L. A. (1976) Nature 261, 68-71. 2. Giudice, G. (1973) Developmental Biology of the Sea Urchin Embryo (Academic Press, New York). 3. Runnstrom, J. (1966) Adv. Morphog. 5,221-325. 4. Hagstrom, B. E. (1956) Ark. Zool. 10, 307-315. 5. Vacquier, V. D., Tegner, M. J. & Epel, D. (1972) Nature 240, 352-353. 6. Veron, M., Foerder, C., Eddy, E. M. & Shapiro, B. M. (1977) Cell 10,321-328. 7. Markman, B. (1958) Acta Zool. (Stockholm) 39, 103-115. 8. Shapiro, B. M. (1975) Dev. Biol. 46,88-102. 9. Runnstr6m, J. L., Monne, L. & Broman, L. (1944) Ark. Zool. 35A, No. 3, 1-32. 10. Ishida, J. (1936) Annot. Zool. Jpn. 15,453-459. 11. Bergmeyer, H. U., Gawehn, K. & Grossl, M. (1974) in Methods of Biochemical Analysis, ed. Bergmeyer, H. U. (Academic Press, New York), pp. 494-495.
Froc.. Natl. Acad. Sa. USA 74 (1977) 12. Inoue, S., Hardy, J. P., Cousineau, G. H. & Bal, A. K. (1976) Exp. Cell Res. 48, 248-251. 13. Lallier, R. (1970) Exp. Cell Res. 63,460-462. 14. Lallier, R. (1971) Experientia 27, 1323-1324. 15. Motomura, I. (1954) Sci. Rep. Tohoku Univ. Ser. 4 20, 219225. 16. Doolittle, R. F. (1973) Adv. Protein Chem. 27, 1-109. 17. Motomura, I. (1954) Sci. Rep. Tohoku Univ. Ser. 4 20, 219225. 18. Dutton, A. & Singer, S. J. (1975) Proc. Natl. Acad. Sci. USA 72, 2568-2571. 19. Andersen, S. 0. (1966) Acta Physiol. Scand. Suppl. 263 66, 1-81. 20. Raven, D. J., Earland, C. & Little, M. (1971) Biochim. Biophys. Acta 251, 96-99. 21. Keeley, F. W. & LaBella, F. S. (1972) Biochim. Biophys. Acta 263,52-59. 22. Kawasaki, H., Sato, H. & Suzuki, M. (1974) Insect Biochem. 4, 99-111. 23. Aeschbach, R., Amado, R. & Neukom, H. (1976) Biochim. Biophys. Acta 439,292-301. 24. Gross, A. J. & Sizer, I. W. (1959) J. Biol. Chem. 234, 16111614. 25. LaBella, F., Waykole, P. & Queen, G. (1968) Biochem. Biophys. Res. Commun. 30,333-338. 26. Himmelhoch, S. R., Evans, W. H., Mage, M. G. & Peterson, E. A. (1969) Biochemistry 8,914-921. 27. Saunders, B. C., Holmes-Siedle, A. G. & Stark, B. P. (1964) Peroxidase-The Properties and Uses of a Versatile Enzyme and of Some Related Catalysts (Butterworths, Washington, DC). 28. Steinhardt, R. A., Epel, D., Carroll, E. J. & Yanagimachi, R. (1974) Nature 252,41-48. 29. Epel, D. (1970) Exp. Cell Res. 61, 69-70. 30. LaBella, F., Keeley, F., Vivian, S. & Thornhill, D. (1967) Biochem. Biophys. Res. Commun. 26,748-753. 31. DeVore, D. P. & Gruebel, R. J. (1977) Fed Proc. 36,679. 32. Petri, W. H., Wyman, A. R. & Kafatos, F. C. (1976) Dev. Biol. 49, 185-199. 33. Katsura, S. & Tominaga, A. (1974) Dev. Biol. 40, 292-297. 34. Roels, F. (1971) Exp. Cell Res. 69,452-456. 35. Klebanoff, S. J. & Smith, D. C. (1970) Biol. Reprod. 3, 236242. 36. Epel, D., Weaver, A. M., Muchmore, A. V. & Schimke, R. T. (1969) Science 163, 294-296. 37. Shaw, P. D. & Hager, L. P. (1961) J. Biol. Chem. 236, 16261630. 38. Smith, D. C. & Klebanoff, S. J. (1970) Biol. Reprod. 3, 229235.
