Peptides from sperm acrosomal protein that initiate egg development

How sperm initiate egg development is being investigated with gametes of the marine worm Urechis. Sperm acrosomal protein, previously shown to activate eggs (Gould et al., 1986, Dev. Biol. 117, 306-318; Gould and Stephano, 1987, Science 235, 1654-1656), was enzymatically cleaved into soluble peptide fragments. When this mixture was added to eggs they activated, and parthenogenetic cleavage often occurred. An active peptide (P23) was purified from the mixture and its sequence was determined to be Val-Ala-Lys-Lys-Pro-Lys. Synthetic peptide had the same biological activity. P23 induced eggs to undergo the complete sequence of changes that normally follows fertilization, including the fertilization potential, completion of meiosis, and DNA replication. When a sperm centrosome was introduced into eggs by prefertilization without activation, and the eggs were subsequently activated by P23, they developed normally to trochophore larvae (the contribution of another sperm component is not ruled out by this experiment). P23 covalently coupled to bovine serum albumin also activated eggs, showing that it acted on the external surface of the egg. The peptide did not activate sea urchin eggs, but did cause oyster eggs to undergo germinal vesicle breakdown.     

Nature of the thiamin-binding protein from chicken egg yolk.

A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.     

Isolation and characterization of thiamin-binding protein from chicken egg white.

A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.     

Purification and identification of antioxidant peptides from egg white protein hydrolysate

Egg white proteins were hydrolysed separately using five different proteases to obtain antioxidant peptides. The antioxidant activity of egg white protein hydrolysates was influenced by the time of hydrolysis and the type of enzyme. Of the various hydrolysates produced, papain hydrolysate obtained by 3-h hydrolysis (PEWPH) displayed the highest DPPH radical scavenging activity. PEWPH could also quench the superoxide anion and hydroxyl radicals, effectively inhibit lipid peroxidation and exhibit reducing power. Then, PEWPH was purified sequentially by ultrafiltration, gel filtration, RP-HPLC and two fractions with relatively strong antioxidant activity were subsequently subjected to LC-MS/MS for peptide sequence identification. The sequences of the two antioxidant peptides were identified to be Tyr-Leu-Gly-Ala-Lys (551.54?Da) and Gly-Gly-Leu-Glu-Pro-Ile-Asn-Phe-Gln (974.55?Da), and they were identified for the first time from food-derived protein hydrolysates. Last, the two purified peptides were synthesized and they showed 7.48- and 6.02-fold higher DPPH radical scavenging activity compared with the crude PEWPH, respectively. These results indicate that PEWPH and/or its isolated peptides may be useful ingredients in food and nutraceutical applications.     

Affinity chromatography of a cysteine proteinase inhibitor from chicken egg protein

A method of affinity chromatography of the inhibitor of cysteine proteinases from chick egg protein using immobilized ficin has been developed. This method yields a highly active inhibitor in an essentially homogeneous state. The molecular weight of the inhibitor is 14,000. The inhibitor suppresses the activity of ficin and papain but produces no effect on the proteolytic activity of trypsin, chymotrypsin, Asp. oryzae serine proteinase or subtilisine. Isoelectric focusing of the inhibitor has revealed the major band with pI 4.35.     

Purification and partial characterization of a cobalamin-binding protein from chicken egg yolk

Cobalamin-binding protein has been purified from chicken egg yolk by using DEAE-cellulose with a NaCl gradient. The resultant protein fraction was subjected to bioaffinity chromatography. The Mr was 38,000 by SDS-PAGE and 39,000 by gel filtration, and indicated that it was a glycoprotein. The Stokes radius was 4.3 nm and the pI 4.1. The protein bound 57CO.B12 with a molar ratio of 1:1 and a Kd of 0.41 microM. The CBP composed 296 amino acids residues. The protein-ligand interaction was inhibited by Cbl analogues.     

Crustacean egg size as an indicator of egg fat/protein reserves

The metabolic properties of crustacean eggs and the major organic reserve utilized for embryogenesis are dependent on the egg-size correlated deposition of fat/protein in the yolk.     

A simplified method for the purification of riboflavin-binding protein from hen's egg yolk

A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.     

Purification and characterization of high antioxidant peptides from duck egg white protein hydrolysates

The hydrolysate from duck egg white protein (DEWP) prepared by “SEEP–Alcalase” at degree of hydrolysis (DH) value of 21% (namely HSA₂₁) exhibited high antioxidant capacity in different oxidation systems. A consecutive chromatographic method was then developed for separation and purification of HSA₂₁, including ion-exchange chromatography, macroporous adsorption resin (MAR) and gel filter chromatography. The final peptides “P_21-3–75-B” were obtained with significantly enhanced antioxidant activity (p < 0.05). It was further confirmed that the product mainly consisted of five oligopeptides (Mr: 202.1, 294.1, 382.1, 426.3, and 514.4 Da). Furthermore, the antioxidant activity of P_21-3–75-B kept stable after in vitro digestive simulation. Antioxidant capacity of the purified peptides was closely related to the molecular mass, hydrophobic amino acid residues, acidic amino acid and some antioxidant amino acids. This research provided a valuable route for producing new natural-source peptides with strong antioxidant capacity and high nutritious value for our daily intake.     

Purification and partial characterization of an actin-like protein from cricket early egg plasmodium

Summary The purification of an actin-like protein from cricket egg yolk plasmodia by different selective extraction procedures, ammonium sulphate precipitation, ion exchange and immunoabsorption chromatography is described. Criteria of purity from analytical ultracentrifugation, SDS-disc electrophoresis, and immunoelectrophoresis are presented. Immunodiffusion analysis was used to control the success of the purification procedures.The molecular weight of the monomeric form is 60000±10%. Polymerization to pearl-chain aggregate structures occurs under different conditions in 0.1 M KCl in the presence of ATP. Vinblastine precipitation leads to similar structures. Possibly related structures and the possible rÔle of this protein in organizing movements in the plasmodial system are discussed.     

Calcium-binding modulator protein from the unfertilized egg of the sea urchin Arbacia punctulata

We have purified and partly characterized a calcium-binding protein from the unfertilized egg of the sea urchin Arbacia punctulata. This protein closely resembles the calcium-binding modulator protein of bovine brain in its molecular weight, electrophoretic mobility, amino acid analysis, and peptide map. It activates bovine brain phosphodiesterase in the presence of calcium but has no effect on the phosphodiesterase of the Arbacia egg. Densitometric scanning of acrylamide gels of arbacia egg homogenates shows the modulator protein to represent 0.1% of the total protein of the egg. At 10(-4) M free calcium, the protein binds four calcium ions per 17,000-dalton molecule. We have used a column of rabbit skeletal muscle troponin-I covalently coupled to Sepharose 4B as an affinity column to selectively purify the Arbacia egg calcium-binding protein. This column has also been used to purify bovine brain modulator protein and may prove of general use in isolating similar proteins from other sources. The technique may be particularly helpful when only small quantities of starting material are available.     

Analysis of egg protein in five carps

Quantitative and qualitative studies of proteins from the mature eggs of five carps—Cirrhinus mrigala, Labeo rohita, Cyprinus carpio, Hypophthalmichthys molitrix, Ctenopharyngodon idella were made. It is found that there is much difference both in the quantity and quality of proteins in these five species. As egg proteins in a species depend on the genome, it is suggested that the study of these protein types may be considered as an additional parameter for characterisation of the fish types.     

Everything from the egg

In 1651, William Harvey opened a real debate when he postulated that the egg is the origin of all life. The central pillar of Harvey's theory sustains that all adults are already present in a smaller size in the egg. The frontispiece of his famous treatise attributed to Richard Gaywood shows Zeus holding an egg that releases small creatures as humans, others animals, and plants. The treatise concludes that no generation occurs without a primordial egg, representing the establishment of ex Ovo Omnia doctrine. Nowadays, evidence supports that both male and female gametes are essential for embryo development following sexual reproduction. Despite obvious differences in morphology and in scale, the oocytes carry out the same role in the development and still demonstrate the mystery of reproduction.     

Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin.

1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.     

An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg

An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.