Isolation and characterization of an ovoinhibitor, a multidomain Kazal-like inhibitor from Turkey (Meleagris gallopavo) seminal plasma.

Turkey seminal plasma contains three serine proteinase inhibitors. Two of them, with low molecular masses (6 kDa), were identified as single-domain Kazal-type inhibitors responsible for regulating acrosin activity. Our experimental objective was to isolate and characterize the inhibitor with the high molecular weight from turkey seminal plasma. The inhibitor was purified using hydrophobic interaction and affinity chromatography. Pure preparations of the inhibitor were used for identification by mass spectrometry, for determination of physicochemical properties (molecular weight, pI, and content and composition of the carbohydrate component), for kinetic studies, and for antibacterial tests. Gene expression and immunohistochemical detection of the inhibitor were analyzed in the testis, epididymis, and ductus deferens. The inhibitor with a high molecular weight from turkey seminal plasma was identified as an ovoinhibitor, which was found in avian semen for the first time. The turkey seminal plasma ovoinhibitor was a six-tandem homologous Kazal-type domain serine proteinase inhibitor that targeted multiple proteases, including subtilisin, trypsin, and elastase, but not acrosin. Our results suggested that hepatocyte growth factor activator was a potential target proteinase for the ovoinhibitor in turkey seminal plasma. The presence of the ovoinhibitor within the turkey reproductive tract suggested that its role was to maintain a microenvironment for sperm in the epididymis and ductus deferens. The turkey seminal plasma ovoinhibitor appeared to play a significant role in an antibacterial semen defense against Bacillus subtilis and Staphylococcus aureus.

Antimicrobial potential of egg yolk ovoinhibitor, a multidomain Kazal-like inhibitor of chicken egg.

Chicken egg ovoinhibitor is a multidomain Kazal-type serine protease inhibitor with unknown function. Comparison of expression between different tissues indicated that ovoinhibitor is highly expressed in the magnum and liver followed by the uterus, which secrete egg white, egg yolk, and eggshell precursors, respectively. The results also revealed that ovoinhibitor expression is increased in the liver during sexual maturation followed by a subsequent decrease in mature hens. Ovoinhibitor was purified from the egg yolk plasma from nonfertilized eggs using two consecutive affinity chromatographies and gel filtration. Purified egg yolk ovoinhibitor was shown to inhibit trypsin and subtilisin. It was shown that purified egg yolk ovoinhibitor exhibited antimicrobial activities against Bacillus thuringiensis . The results suggest that this anti-protease plays a significant role in antibacterial egg defense against Bacillus spp., preventing contamination of table eggs (nonfertilized eggs) and protecting the chick embryo (fertilized eggs).

Comparison of extraction conditions for milk and hen's egg allergens.

The evaluation of recovery rates by extracting milk powder and egg powder using eleven different extractants gave approximately similar results for both foods. Compared with the other extraction solutions investigated, '1% Tween 20® and 0.4% Triton X-100®' and '4% SDS' are the most suitable extractants to isolate proteins of hen's egg or milk. When comparing calculated protein recovery rates of egg and milk powder extracts, the results clearly indicated that the choice of a suitable extractant is of particular importance. Qualitative investigation of the extracts via LDS-PAGE followed by silver staining as well as immunoblotting confirmed the results of protein quantification. Hence, the immunoblots showed that the extraction agents had no negative influence on the antigenicity of the extracted allergenic proteins. In this study, variation of extraction temperature led neither to any benefit in extraction quality nor to degradation. Changing pH did not reveal any trends, but progressive protein hydrolysis under strong alkaline conditions. Evaluation of recovery rates as well as results of unspecific and specific staining of the extracts showed that an extraction time of 1 h is sufficient for an appropriate sample preparation. For investigations with and without food matrix different results were obtained. In summary, wheat starch did not influence the extraction quality within all examined materials and different extractants. In contrast, using fat powder and dry cake mix, respectively, led to different results in the extraction procedure. When fat powder and dry cake mix were used as food matrices, some protein recovery rates decreased and some increased depending on the allergen material. These results highlight the fact that the suitability of the extractant not only depends on the properties of the allergen but furthermore on the type of matrix containing the allergen.

In vitro determination of the allergenic potential of technologically altered hen's egg.

Hen's egg allergy represents one of the most common and severe IgE-mediated reactions to food in infants and young children. It persists, however, in many cases also lifelong. Therefore, the aim of this study was the detailed analysis of a technological process used to reduce the allergenic potential of hen's egg. The investigation focused on the pasteurized egg as starting material, intermediate, and final products of a nine-step manufacturing process performed for use of eggs in convenience products appropriate for allergic individuals. The steps consisted of a combination of various heat treatments and enzymatic hydrolyses. The alterations were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, enzyme allergosorbent test (EAST) inhibition, and mass spectrometry. Thereby it could be demonstrated that the allergenic potential of the raw material was reduced from step to step, and despite the known stability against heat and proteolysis of certain egg proteins, the total allergenic potential was finally below 1/100 that of the starting material without a significant change in texture and flavor as evaluated in various products.

High-capacity purification of hen egg-white proteins by ion-exchange electrochromatography with an oscillatory transverse electric field.

The ion-exchange electrochromatography with an oscillatory electric field perpendicular to the mobile-phase flow driven by pressure (pIEEC) was used to separate hen egg-white (HEW) proteins. The results were compared with those of normal ion-exchange chromatography (IEC). The column was designed as three-compartment rectangular column of 2-mL with dimensions (length x width x depth) of 40 x 10 x 5 mm(3) and the electric field was applied across the direction of column width. Q Sepharose FF was packed into the central compartment as the chromatographic bed. It was confirmed that the dynamic binding capacity (DBC) of different proteins (ovotransferrin and ovalbumin) in the HEW solution increased 2.3 times when an oscillatory electric current of 30 mA at 1/20 Hz was applied in the transverse column direction. Then, the HEW proteins were separated by the pIEEC at loading amounts 2.3-fold higher than those by the IEC. When the feedstock of about one-third of the DBC was applied to the columns (i.e., 7 mL for the pIEEC and 3 mL for the IEC), similar separation efficiencies of the two chromatographic modes were achieved. Both the recovery yield and purity reached 73% to over 90%. The results indicate that the pIEEC is promising for high-capacity purification of proteins.

Characterization of lipovitellin in eggs of the polychaete Neanthes arenaceodentata.

The ooplasm of mature oocytes of the polychaete Neanthes arenaceodentata is characteristically filled with yolk platelets. A major component of these structures is lipovitellin, which provides energy and materials required by newly hatched larvae. The lipovitellin isolated and purified from the fertilized eggs of this polychaete was a high-density lipoprotein composed of protein (57%), lipid (42%) and carbohydrate (1%). The lipid component included phospholipids (92% of lipid), triacylglycerol (3% of lipid) and cholesterol (3% of lipid), while sodium dodecyl sulfate-gel electrophoresis showed the major protein component was a 120-kDa peptide. Microscopically, mature oocytes were present in the coelom along with phagocytic eleocytes. The presence of muscle fragments and oil droplets in eleocytes suggests that eleocytes play an important role in providing the protein and lipid needed for the assembly of lipovitellin in the oocytes.

Limited proteolysis and biophysical characterization of the lipovitellin homology region in apolipoprotein B.

Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.

Significant modification of lipid metabolism in aged persons following the treatment with a nutritive supplement containing embryonary peptides--preliminary results.

HUMANOFORT is a nutritive supplement extracted and purified from embryonated chicken eggs according to an original procedure under licence. Humanofort received the suitable consent from the Romanian Ministry of Health. The main components of Humanofort are two oligopeptides of 5,000 and 10,000 D molecular weight. 40 subjects aged 50-75 years (18 men and 22 women) consumed, daily, 4 caps of Humanofort for 60 days. The samples of blood from each subject were obtained before and after treatment. Therefore, each subject was his own control. In all subjects, after treatment, total cholesterol and LDL-cholesterol decreased approximately 30% as compared with the initial values. In 80% of patients, an increase of HDL cholesterol and a decrease of the insulin level in blood were also observed. After treatment, the cardiac risk factors (Aethna 2000 Program), such as total cholesterol/HDL and Apolipoproteins B/A were shifted towards lower range. These long-lasting modifications have an adaptative-regulatory character and seem to be produced by the growth factors contained in Humanofort.

Ovoinhibitor in the chicken bursa of Fabricius: identification, isolation, and localization.

A monoclonal antibody (Mab) developed against a partially purified bursal protein extract was found to bind specifically to a single cell type in the cortico-medullary border region of the chicken bursa of Fabricius. These cells were microscopically similar to the bursal secretory dendritic-like cells. A product with an apparent molecular weight of approximately 56 kDa on SDS-polyacrylamide gel electrophoresis was immunopurified from bursal extracts by utilizing this Mab. This product was subjected to peptide digestion and protein sequencing. The two resulting sequences perfectly matched the known sequence of chicken ovoinhibitor. Gene-specific polymerase chain reaction (PCR) primers were designed for the ovoinhibitor, RNA was purified from chicken bursae, and reverse transcription/PCR was performed. Two amplicons with the expected size for ovoinhibitor mRNA were obtained. These data suggest that the gene for ovoinhibitor is expressed in the bursa of Fabricius, and that the bursal secretory dendritic-like cells may be a previously unreported source of ovoinhibitor.

Expression and characterization of chicken ovoinhibitor in Pichia pastoris.

Chicken ovoinhibitor cDNA was prepared by reverse transcriptase-polymerase chain reaction (RT-PCR) using chicken oviduct mRNA. The ovoinhibitor cDNA was successfully cloned downstream from the AOXI promoter of pPICZalphaA plasmid vector to facilitate its expression in the methylotrophic yeast Pichia pastoris. The pPICZalphaA carrying the ovoinhibitor cDNA was integrated into the Pichia genome. The secreted recombinant ovoinhibitor was purified by ion-exchange chromatography on a DEAE sepharose column. The recombinant ovoinhibitor had a molecular mass of 49 kDa, as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and time of flight-mass spectrometry (TOF-MS) analyses. The recombinant ovoinhibitor, just as the native ovoinhibitor, showed inhibitory activity against trypsin, chymotrypsin and elastase.

Development of a sandwich enzyme-linked immunosorbent assay for the detection of egg residues in processed foodst.

Chicken eggs are used extensively as an excellent source of dietary proteins. These proteins have many functional properties, making them valuable food ingredients. However, eggs are a frequent cause of food hypersensitivity, especially in children. Of major concern to food processors is the inadvertent cross-contact of food products with allergenic residues, which could result in potentially life-threatening reactions in those with a food allergy. The aim of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of undeclared egg residues in foods. Commercially purified ovalbumin (OVA) and dehydrated egg white solids were used as antigens to induce antibodies in rabbits and goats. Reference pasta standards and various food samples were extracted, then clarified by centrifugation. Goat anti-egg white antibodies were used as the capture reagent, nonspecific sites were blocked with gelatin, then standard and sample extracts were added. Rabbit anti-OVA antibodies were used as detector antibodies, followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. Twenty brands of egg-free pasta (two lots each) were analyzed using the ELISA. Fourteen common pasta ingredients were also evaluated for cross-reactivity problems in the method. The detection limit of the assay was 1 ppm spray-dried whole egg. Fifty-five percent (22 samples) of the egg-free pasta samples tested positive for the presence of undeclared egg residues, with values ranging from 1 to >100,000 ppm. Minimal cross-reactivity was encountered in general, but portobello mushrooms and basil caused some minor matrix effects. This sandwich-type ELISA method can be used to detect undeclared egg residues in processed foods and to evaluate industrial clean-up operations.

Development of an ELISA for vitellogenin in whole body homogenate of zebrafish (Danio rerio).

The yolk protein, lipovitellin (Lv) was purified from ovaries of mature female zebrafish (Danio rerio) by gel filtration and anion exchange chromatography. Polyclonal antibodies against Lv were raised in rabbits. Anti-Lv IgG was purified by affinity chromatography. SDS-PAGE followed by Western blotting was performed to analyse the specificity of the antibody and the immunological similarities between Lv and vitellogenin (Vtg). Anti-Lv IgG was used to develop a direct non-competitive sandwich ELISA to measure Vtg concentrations of whole body homogenate (WBH) in zebrafish. The intra- and interassay variabilities were 5.8% and 10.4%, respectively. The sensitivity was 0.2 ng Vtg x ml(-1) and the practical detection limit was 40 ng Vtg x g(-1) fish (wet weight). Adult male zebrafish were exposed to a nominal water concentration of 10 ng x l(-1) of ethinylestradiol (EE2) in a semi-static exposure system for 7 days. Compared with the control group, exposure to 10 ng EE2 x l(-1) induced a 200-fold increase in Vtg levels.

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.

Two forms of vitellogenin, yielding two distinct lipovitellins, play different roles during oocyte maturation and early development of barfin flounder, Verasper moseri, a marine teleost that spawns pelagic eggs.

Two forms of vitellogenin (Vg), Vg A and Vg B, were identified in serum from estrogen-treated barfin flounder (Verasper moseri). Structural changes of lipovitellins (Lvs) derived from the two Vgs were examined during vitellogenesis and oocyte maturation. Two Lvs, vLv A and vLv B, were identified electrophoretically and immunologically in postvitellogenic oocytes. Each appeared to be composed of distinct heavy chains (vLvH A, M(r) 107,000, and vLvH B, M(r) 94,000) and light chains (vLvL A, M(r) 30,000, and vLvL B, M(r) 28,000) when analyzed by SDS-PAGE. Results from N-terminal amino acid sequencing and Western blotting using antisera to vLvH A and vLvH B verified that there are two Vg polypeptides in serum from estrogen-treated fish, Vg A (M(r) 168,000) and Vg B (M(r) 175,000), which give rise to vLvH A-vLvL A and vLvH B-vLvL B, respectively. N-terminal sequencing revealed two sequences for both phosvitin and beta'-component, supporting the concept of duality for all three classes of Vg-derived yolk proteins. During oocyte maturation, native dimeric vLv B was dissociated into a native M(r) 170,000 monomer (oLv B). Meanwhile, vLv A was extensively cleaved including complete degradation of vLvH A into free amino acids. We propose that the quantitative ratio of vLv A to vLv B in postvitellogenic oocytes regulates the buoyancy of the spawned pelagic eggs by controlling availability of free amino acids which function as osmotic effectors during oocyte hydration. The vLv A/vLv B ratio likely also controls the proportional availability of different types of nutrients, free amino acids versus Lv, for use during embryonic development.

Isolation and characterization of domain I of ovoinhibitor.

Domain I of ovoinhibitor was isolated by subjecting the protein to specific chemical cleavage by cyanogen bromide followed by repeated gel filtration. The first domain of ovoinhibitor was found to be homogeneous by the criteria of gel chromatography, SDS-PAGE and PAGE. Mr values by gel filtration (10900) and SDS-PAGE (8300) were slightly higher than that computed from amino-acid sequence. This discrepancy has been attributed to the glycoprotein nature of domain I as it was found to contain 10% neutral carbohydrate and 2% sialic acid. Fluorescence spectral properties showed the presence of tryptophan in domain I. The amino-acid composition of domain I isolated in this study was in very good agreement with that computed from amino-acid sequence. Gel filtration behaviour of the first domain was consistent with a Stokes radius of 1.6 nm and a frictional ratio of 1.2 suggesting asymmetry and/or excessive hydration. Domain I was found to be a potent inhibitor of bovine trypsin but was virtually devoid of activity against chymotrypsin, elastase and proteinase K. The equilibrium association constant for domain I-trypsin complex was computed to be 6.6x10(8)M-1.

Vitellogenin and lipovitellin: zinc proteins of Xenopus laevis oocytes.

Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (pK 4.8). Once internalized by receptor-mediated endocytosis, vitellogenin is cleaved into multiple polypeptides, i.e., the two lipovitellin subunits (1 and 2) plus phosvitin; these are then stored as microcrystals within yolk platelets. We here show by metal analysis of the individual proteins generated by vitellogenin processing that zinc and calcium occur in different domains of the vitellogenin polypeptide chain. All of the vitellogenin zinc is present in lipovitellin, in amounts equal to 1 mol of zinc/141 kDa. Calcium, in contrast, is detected exclusively in phosvitin which, in addition, contains 3 mol of magnesium/35 kDa, apparently acquired following vitellogenin entry into the oocyte. The zinc in lipovitellin is removed by OP in a concentration-dependent manner with a pK of 4.8, identical to that obtained for vitellogenin, and by exposure to acidic conditions (below pH 5). Following removal of zinc, the two lipovitellin subunits remain associated, suggesting that zinc is not involved in their interaction. On exposure to 1% SDS, lipovitellin does dissociate into 106 and 33 kDa subunits. The presence of stoichiometric quantities of zinc in both vitellogenin and lipovitellin calls for the study of the hitherto unrecognized biochemistry and functions of these proteins in zinc metabolism and development of the frog oocyte and embryo.

Purification of egg-white allergens.

Purification procedures for the four egg-white proteins ovomucoid, ovotransferrin, ovalbumin, and lysozyme are presented with reference to mechanistic studies at epitope levels of allergic reactions to these proteins. The applied procedures resulted in four preparations containing less than 0.1% contaminating proteins each. The purified protein preparations were characterized by SDS-PAGE and by crossed immunoelectrophoresis with polyclonal antibodies raised against an egg-white extract or the purified proteins. The necessity of these well-characterized proteins in studies on allergic reactions was shown by testing human sera in immunoblots of lysozyme, and by immunoblots of ovomucoid probing with antibodies against the proteins.

Purification and characterization of vitellogenin and lipovitellins of the sand crab Emerita asiatica: molecular aspects of crab yolk proteins.

In the mole crab Emerita asiatica, the main yolk proteins consist of two slow moving lipovitellins (Lv I and Lv II) of glycolipoprotein nature. Lv I cleaves into subunits (MW: 109,000 and 105,000) and Lv II gives rise to six subunits (MW: 65,000, 54,000, 50,000, 47,000, 44,000, and 42,000) in SDS-PAGE (with beta-mercaptoethanol). In order to observe the stability of Lv II as well as to achieve better resolution of the proteins, two different buffer systems (Phosphate buffered saline and tris-buffered saline), 40% sucrose, and glass distilled water were used as homogenizing media. Among them, better resolution was achieved with tris-buffered saline and 40% sucrose, and tris-buffered saline seems to be the ideal medium for elution of Lv II. The analysis of biochemical constituents of the major Lv II reveals a percentage composition of 69.325, 27.927, and 2.753 respectively for protein, lipid, and bound sugars. In the I stage embryo, protein comprises about 67.276%, lipid 29.65%, and bound sugars 3.015%. Vitellogenin (Vg) electrophoretically corresponding to the Lv I and Lv II was present in the female haemolymph during the entire period of embryogenesis. The number of subunits (8) of Vg in all stages remained unaltered and their approximate molecular weights were Vg1, 91,000; Vg2, 87,000; Vg3, 83,000; Vg4, 61,000; Vg5, 58,000; Vg6, 45,000; Vg7, 42,000; and Vg8, 38,000. Different proteins present in the embryos (I and IV stage) and the serum obtained from the animal carrying the I stage embryo were separated by gel-filtration in high performance liquid chromatography (HPLC). Sephadex (G-200) gel filtration chromatography was used to purify the Lv II in large quantity. Total lipid extracted from Lv II as well as the embryos belonging to different stages of development were separated into their constituent neutral, glycolipids, and phospholipids, using silicic acid column chromatography. Thin layer chromatography (TLC) was used to isolate the different phospholipids purified from various stages of embryos and Lv II. As many as seven different phospholipids were separated from Lv II and I and IX stage embryos; and whereas thin layer chromatogram of V and VI stage embryos showed six different phospholipids, embryos of VII and VIII stage contained four phospholipid species. Cholesterol, glycolipids, and individual phospholipids isolated from the Lv II and I stage embryo were quantified spectrophotometrically and the results were discussed.

Ovostatin, an endogenous trypsin inhibitor of sea urchin eggs: purification and characterization of ovostatin from eggs of the sea urchin, Strongylocentrotus intermedius.

A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K-21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC50 (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 +/- 0.13 micrograms/ml (4.55 +/- 0.65 x 10(-8)M), 3.0 +/- 0.28 microgram/ml (1.5 +/- 0.14 x 10(-7)M), and 4.8 +/- 0.2 microgram/ml (2.4 +/- 0.1 x 10(-7)M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025-0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca2+ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization.