Preparation, Properties, and Composition of Nereis vitellin, the Yolk Protein of the Annelid, Nereis virens

In the literature, oogenesis in nereid annelids is considered to be a model system because, unlike other system studied, nereid oocytes are thought to synthesize the bulk of their yolk protein themselves. As the first step to test the validity of this hypothesis, nereid yolk protein was characterized biochemically. Vitellin, the main fraction of the soluble yolk proteins, was prepared from Nereis virens oocytes. Preparation, purification, and some physical characteristics of this green-colored protein Nereis vitellin are described. The molecular weight was determined by gel chromatography as 420,000 daltons. With regard to the amino acid composition, Nereis vitellin was found to resemble both insect vitellins and an average protein, as defined by other authors. Methionine and cysteine were found in traces only. By staining procedures, Nereis vitellin was characterized as lipoglycoprotein. Nereis vitellin was also prepared from the coelomic fluid of gravid females of Nereis virens .     

Purification on Renografin Density Gradients of Chlamydia trachomatis Grown in the Yolk Sac of Eggs

Chlamydia trachomatis grown in the yolk sac of embryonated eggs was purified by centrifugation on continuous isopycnic Renografin density gradients. A band of chlamydial particles with a buoyant density of 1.20 contained 70% of the starting particles, and electron microscopy revealed the virtual absence of contaminating egg material. Centrifugation on Renografin gradients caused only a moderate decrease in infectivity. For large-scale purification, infected yolk sac was centrifuged through Renografin solutions, resulting in greater than 60% recovery of starting chlamydial particles, but less than 1% recovery of the dry weight and protein.     

Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat

The yolk sac of the fetal rat and the proximal small intestine of the neonatal rat selectively transport maternal IgG. IgG-Fc receptors are thought to mediate transport across the epithelium of both tissues. We used a mouse mAb (MC-39) against the 45-54-kD component of the Fc receptor of the neonatal intestine to find an antigenically related protein that might function as an Fc receptor in fetal yolk sac. In immunoblots of yolk sac, MC-39 recognized a protein band with apparent molecular mass of 54-58 kD. MC-39 bound to the endoderm of yolk sac in immunofluorescence studies. In immunogold-labeling experiments MC-39 was associated mainly with small vesicles in the apical cytoplasm and in the region near the basolateral membrane of endodermal cells. The MC- 39 cross-reactive protein and beta 2-microglobulin, a component of the intestinal Fc receptor, were copurified from detergent-solubilized yolk sac by an affinity purification that selected for proteins which, like the intestinal receptor, bound to IgG at pH 6.0 and eluted at pH 8.0. In summary, the data suggest that we have isolated the Fc receptor of the yolk sac and that this receptor is structurally and functionally related to the Fc receptor of the neonatal intestine. An unexpected finding is that, unlike the intestinal receptor which binds maternal IgG on the apical cell surface, the yolk sac receptor appears to bind IgG only within apical compartments which we suggest represent the endosomal complex.     

The purification of IgY from chicken egg yolk by preparative electrophoresis

Chicken IgY has been purified from egg yolk by preparative electrophoresis on the Gradiflow, a system which has been employed for the purification of a wide range of proteins with high recovery and biological activity. Protein purification on the Gradiflow utilises electrophoresis with selected combinations of porous membranes and buffers. The purification of IgY was achieved by initial PEG lipid precipitation, then a single step Gradiflow run by a strategy based on the relatively high pI range of IgY compared to other egg yolk proteins. The IgY yields obtained from the delipidised supernatant are consistently greater than 80% by immunoassay. The purity of the IgY fraction compared favourably with IgY prepared using three commercial products.     

An improved method for isolation of anti-viper venom antibodies from chicken egg yolk

The production of antibodies and its purification from mammalian blood has been found low yielding and laborious. Therefore, anti snake venom antibodies for therapeutic use is obtained mostly as polyvalent whole serum or partially purified polyvalent immunoglobulin. The side effects of anti snake venom (ASV) therapy are mainly serum sickness and renal failure, which may be reduced by using sufficiently pure antibodies. Therefore, we have standardized a simple method for production of purified antivenom. Here, we present the development of polyclonal antibodies against viper venom in hens and its isolation from the egg yolk of immunized birds. We have modified the reported methods of purification of immunoglobulin from egg yolk, and thus yielded 90% purity of the protein. The modified method involves only two steps, such as removal of lipids from the diluted egg yolk by a freeze-thaw cycle and centrifugation, followed by gel filtration on Biogel P-150. The advantages are that the process is very simple, and from one egg, 100+/-20 mg of pure immunoglobulin is obtained. The antibodies are present in the egg for up to 100 days after the immunization. Thus, using small amounts of venom, a large quantity of the immunoglobulin is obtained in a sufficiently pure form. The antigen binding ability of the pure antibody is found good by the Ouchterlony's double diffusion experiment.     

Functional reconstitution of the partially purified aspartate-glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria has been solubilized with detergent. The transport protein was partially purified by chromatography on hydroxyapatite in the presence of dodecyl octaoxyethylene ether and high concentrations of ammonium acetate. During purification, the aspartate/glutamate carrier was identified by functional reconstitution into egg yolk phospholipid liposomes. After hydroxyapatite chromatography the protein is 30 fold enriched in aspartate/glutamate transport activity but still contains ADP/ATP-carrier and phosphate carrier. The reconstituted activity is specific for exchange of L-aspartate and L-glutamate and is similar to intact mitochondria with respect to substrate affinity and inhibitor sensitivity.     

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.     

Egg yolk phosvitin: preparation of metal-free purified protein by fast protein liquid chromatography using aqueous solvents

Two chromatographic methods for hen egg yolk phosvitin purification avoiding organic solvents were evaluated. Hydrophobic interaction and ion-exchange chromatographies were applied to isolated phosvitin. Hydrophobic interaction chromatography has better capacity than ion-exchange chromatography to fractionate phosvitin in their different polypeptides, but its protein yield was lower (0.7 vs. 1.7% of egg yolk dry matter). Finally, ion-exchange chromatography was selected and allowed to fractionate phosvitin polypeptides, including the recovering of phosphoproteins with high electrophoretic mobility: phosvettes. Highly purified (>98%) and free metal protein was obtained in reduced time. Phosvitin polypeptide heterogeneity was evidenced.     

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.     

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.     

Purification and characterization of biotin-binding protein II from chicken oocytes.

BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.     

Chicken egg yolk stabilizes the reverse transcriptase activity in type C particles produced by cultured MOPC-315 murine myeloma cells.

Type C particles released from cultured murine myeloma MOPC-315 cells were significantly protected when the purification steps were all conducted in the presence of 10% chicken egg yolk fluid. The yolk fluid also slowed down the inactivation of viral particles during incubation at 37 degrees C and enabled full recovery of viral particles through several cycles of freezing and thawing. The purification of viral particles in the presence of yolk fluid did not affect the capability of the viral DNA polymerase to reverse-transcribe the virion RNA in vitro, nor that of the viral RNA to act as a functional template.     

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."     

Purification of adenosine deaminase from chicken-egg yolk by affinity column chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

Purification of Adenosine Deaminase from Chicken-Egg Yolk by Affinity Column Chromatography

Adenosine deaminase (adenosine aminohydrolase; E.C. 3.5.4.4) has been purified 4686-fold from egg yolk. The procedure developed was used to isolate the enzyme from eight chicken eggs. An easily prepared affinity column employing purine riboside was used as the final step in the purification. The method developed permits the rapid isolation and a high recovery of the protein. The specific activity of the enzyme preparation obtained is 81.4 mU/mg.     

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.     

Vitamin D-dependent calcium-binding protein of mouse yolk sac. Biochemical and immunochemical properties and responses to 1,25-dihydroxycholecalciferol

The present studies were performed to further characterize a mouse yolk sac protein which is similar or identical to the vitamin D-dependent intestinal calcium-binding protein (CaBP). Yolk sac protein and purified rat intestinal CaBP displayed full identity upon immunodiffusion (Ouchterlony) using antiserum to the rat intestinal CaBP. Immunoreactive CaBP in yolk sac homogenates eluted from gel permeation columns with the low molecular weight peak of 45Ca2+ binding (Chelex assay), and the electrophoretic mobility of the protein was markedly increased by EDTA. On days 11-13 of gestation, the concentrations of immunoreactive CaBP in yolk sac were 4-5-fold higher than in placenta; by days 16-17, the concentrations in yolk sac and placenta were similar. Incubation of yolk sac with [3H]leucine demonstrated synthesis of immunoprecipitable [3H]CaBP. A single band of 3H-labeled protein was seen on sodium dodecyl sulfate gel electrophoresis of the immunoprecipitate. This protein co-migrated with radioactive placental CaBP with an apparent Mr of 10,050. Addition of 1,25-dihydroxycholecalciferol (calcitriol) to organ culture media with or without serum increased the amount and concentration of CaBP in yolk sac (p less than 0.001) at 48 h. CaBP synthesis in yolk sac appeared to be independent of calcitriol concentrations in the maternal circulation since injection of the hormone into the maternal compartment produced no change in yolk sac CaBP despite increases of maternal intestinal and renal CaBP. These studies demonstrate that yolk sac immunoreactive CaBP is synthesized in yolk sac and has an apparent molecular size and calcium-binding properties characteristic of mammalian vitamin D-dependent calcium-binding proteins. The in vitro response of yolk sac CaBP to calcitriol is the first evidence of a vitamin D effect on the fetal membranes and suggests one function for calcitriol receptors in these tissues.     

莫桑比克非鲫卵黄形成的电镜观察

运用透射电镜观察了莫桑比克非鲫卵母细胞的生长.根据卵母细胞的大小和内部结构特征,将其分为四个时期:卵母细胞生长早期:卵黄泡形成期:卵黄积累期:卵黄积累完成期.本文着重研究了主要卵黄成分--卵黄球的形成过程.卵黄球属外源性卵黄,由卵母细胞通过微胞饮作用吸收肝脏合成的卵黄蛋白原后形成的.在卵黄大量积累前,卵母细胞内的线粒体和多泡体聚集成团,构成卵黄核,继而线粒体大量增殖,线粒体形状发生改变,形成同心多层膜结构,为大量的卵黄物质积累提供场所.最终形成的卵黄球由被膜、卵黄结晶体和两者之间的非结晶区三部分组成.       

The origin of protein and fatty yolk in Rana pipiens. III. Intramitochondrial and primary vesicular yolk formation in frog oocytes.

The precise origin of the primary yolk precursor complex or primary vesicular yolk is obscure but in its earliest recognizable stage it is a typical multivesicular body which first acquires a moderately electron-dark matrix. Following this, an extremely electron-dark amorphous material, the yolk protein, appears within the precursor. This yolk protein increases in amount as the yolk vesicle grows and by the time the precursors are about 1 micrometer in diameter this protein is partly to almost completely crystalline. Yolk originating within mitochondrial cristae unlike that in the yolk precursor complexes is crystalline from its earliest appearance. Intracristae mitochondrial yolk crystals have a spacing of 70--85 A. Their molecular organization appears in some sections as electron-dark lamellae and in others as light cylinders surrounded by an electron-dark matrix.