Fatty acid composition and thermal behavior of natural sphingomyelins.

We found significant differences in the fatty acid composition of several bovine brain, egg yolk and sheep erythrocyte sphingomyelins. These differences in fatty acid composition influence the thermal behavior of hydrated sphingomyelin as recorded by differentail scanning calorimetry. Significant differences were also found in the temperature and complexity of the order-disorder phase transitions of bovine brain sphingomyelin obtained from different sources which, in general, correlate with the relative content of the saturated fatty acids (palmitic (C16:0) and stearic acid (C18:0) acids) and the long unsaturated nervonic acid (C24:1).

31P-NMR characterization of hen egg yolk and egg white.

The avian egg is an isolated system where embryonic development can easily be studied. Furthermore, the system can be subjected to various environmental constraints. Its size is such that it can easily be accommodated in most NMR instruments. This is demonstrated by a recent 31P-NMR surface coil experiment where the increase of embryo size and development could be judged by the levels of ATP and phosphocreatine (Belton, P.S., Gordon, R.E., Jones, J.M. and Shaw, D. (1983) Br. Poult. Sci. 24, 429-433).

Isolation and characterisation of a biotin-binding protein from the pregnant-rat serum and comparison with that from the chicken egg-yolk.

A biotin-binding protein exhibiting partial immunological cross-reactivity with the purified chicken egg-yolk biotin-binding protein has been detected, for the first time, in the sera of pregnant/estrogenised female rats but not of the normal males. This protein, purified by affinity chromatography on a biotin-AH-Sepharose was homogeneous by electrophoretic and immunological criteria. It was a glycoprotein of Mr 66,000 without any detectable subunites, had a pI 4.1, and specifically bound [14C]biotin. Several structural and functional features of the biotin-binding protein of rat and chicken were found to be similar. These included immunological cross-reactivity, acidic and glycoprotein nature, ability to tightly bind [14C]biotin, estrogen stimulation for their appearance in circulation, and the pattern of distribution of radioiodinated peptides upon proteolysis with trypsin.

Characterization of complexes of egg yolk phosphatidylcholine and apolipoprotein A-II prepared in the absence and presence of sodium cholate.

Complexes of apolipoprotein A-II and egg yolk phosphatidylcholine were prepared in mixtures of different composition in the absence and presence of sodium cholate. By gradient gel electrophoresis, complex preparations were polydisperse and particle size distributions were influenced by the composition of the reconstitution mixture. Complexes generally exhibited a discoidal morphology by electron microscopy, but showed increased formation of vesicular complexes at elevated levels of egg yolk PC in the mixtures. By chemical crosslinking, complexes formed in the absence of cholate were shown to consist primarily of discoidal species with three apolipoprotein A-II molecules per particle in the mixtures investigated; complexes formed in the presence of cholate included species ranging from three to five apolipoprotein A-II per particle. The number of apolipoprotein A-II per particle and the sizes of the complexes, prepared in cholate, increased with increase of egg yolk PC in the reconstitution mixture. Relative to the particle size distribution of discoidal complexes formed in the absence of cholate, those prepared in cholate showed a distribution shifted to larger particle sizes. Complexes of similar particle size distribution formed in the presence or absence of cholate showed similar physical-chemical properties. Discoidal complexes with the same number of apolipoprotein A-II per particle but of different size and composition were observed, suggesting the possibility of some conformational adaptation of apolipoprotein A-II leading to stabilization of egg yolk PC bilayers of different diameter. Properties of particle size distributions of discoidal complexes prepared in cholate of apolipoprotein A-II and egg yolk PC were compared with those of complexes of apolipoprotein A-I previously reported (Nichols, A.V., Gong, E.L., Blanche, P.J. and Forte, T.M. (1983) Biochim. Biophys. Acta 750, 353-364).

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.

Hen's egg yolk cholinesterase. Purification, characterization and comparison with hen's liver and blood plasma cholinesterase.

The cholinesterase (acylcholine acylkhydrolase, EC 3.1.1.8) of chicken egg yolk was partly purified and characterized. It was compared to homologous enzymes of liver and blood plasma of laying hens. During gel filtration, yolk and liver cholinesterase were resolved into two fractions. Blood plasma cholinesterase showed one form only, identical with yolk and liver cholinesterase 1 *** (EC 3.1.1.8). This form has an Mr of 440 000 and may be a tetramer of a cholinesterase form present in yolk and liver (Mr 104 000). Substrate specificity, pH optima, Km values, the influence of effectors (ammonium derivatives, choline, eserine, fluoride), gel filtration, gel electrophoresis, isoelectric focusing and affinity chromatography, all point to a very close similarity, if not identity, of the corresponding forms.

Purification of biotin-binding protein from chicken egg yolk and comparison with avidin.

A simple alternative procedure for the purification in higher yields of the biotin-binding protein from the chicken egg yolk in a ligand-free form is described. The isolated protein was homogeneous by the criteria of polyacrylamide gel electrophoresis, gel filtration chromatography, immuno-double diffusion and immuno-electrophoresis. The protein had an Mr of 72 000 +/- 2000 and was a homotetramer of subunit Mr of 18 000 +/- 1000. It bound [14C]biotin in the molar ratio of 1:4 with an association constant (Ka) of 0.58 X 10(12) M-1. The yolk biotin-binding protein and avidin exhibited qualitatively similar spectral changes on interaction with biotin and p- hydroxyazobenzoic acid, but quantitatively these changes were more pronounced with avidin. Despite the lack of gross immunological cross-reactivity between the two biotin-binders, evidence based on immunological techniques for some degree of common conformational characteristics restricted to or around the ligand-binding sites of the two proteins was adduced. The mixed subunits of the two proteins failed to form hetero-oligomers on reconstitution.

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes.

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.

Stimulation of the hormone-sensitive triacylglycerol lipase from adipose tissue by phosphatidylethanolamine.

The activity of a pigeon adipose tissue hormone-sensitive triacylglycerol lipase preparation was increased from 2- to 5-fold by the presence of phosphatidylethanolamine in assays with three different methods of preparing triolein substrates. Phosphatidylethanolamine from egg yolk produced the greatest stimulation of lipase activity; the stimulation was concentration-dependent but was not time-dependent. A comparable increase in triacylglycerol lipase activity due to phosphatidylethanolamine was also observed with enzyme preparations from chicken and rat adipose tissue. Phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, Triton X-100 and sodium dodecyl sulfate all inhibited enzyme activity. Phosphatidylethanolamine had no effect on acid lipase activity in the pigeon adipose tissue preparation. Preincubation of the pigeon adipose tissue lipase with ATP, cyclic AMP and protein kinase resulted in a 2.15-fold activation of hydrolase activity determined in the absence of phosphatidylethanolamine. In contrast, non-activated and protein kinase-activated forms of the lipase were characterized as having very nearly the same activity in assays with substrate preparations containing phosphatidylethanolamine. The phosphatidylethanolamine-dependent stimulation of lipase activity was characterized kinetically as being due to an increase in maximal velocity. The modulation of the adipose tissue hormone-sensitive lipase activity by phospholipids could be involved in the hormonal regulation of lipolysis.

Cleavage of two yolk proteins from a precursor in Caenorhabditis elegans.

Four yolk proteins have been identified previously in the nematode Caenorhabditis elegans. However, only two of these proteins ( yp170A and yp170B ) are found among the products of in vitro translation of nematode RNA. The other two yolk proteins ( yp115 and yp88 ) are apparently cleaved from a precursor polypeptide of approximately 180,000 Mr. This precursor has been identified as an in vitro translation product and as a metabolically unstable polypeptide in vivo. It is bound by immunoglobulin G (IgG) specific for yp115 and by IgG specific for yp88 . The immunoadsorbed material yields the same pattern of fragments on partial digestion with Staphylococcus aureus V8 protease regardless of whether anti- yp115 or anti- yp88 IgG is used in the adsorption. Like the yp170 polypeptides, the yp115 / yp88 precursor is synthesized by the intestine and secreted intact. The precursor is evidently cleaved to yield yp115 and yp88 after secretion from the intestine but independent of the presence of the gonad. Thus, cleavage probably occurs in the body cavity of the nematode.

A new embedding medium for cryo-sectioning eggs of high yolk and lipid content.

Eggs can be cryo-sectioned without rupture of the yolk- and lipid-containing areas after embedding in a mixture of polymerized acrylamide, crosslinked gelatine, 'Jung' medium and buffer. For initial fixation of the section on the slide a fixative containing glue is employed, followed by postfixation treatment. The new technique improves sectioning of large eggs including embryonic stages for histological and histochemical studies. It is especially useful for eggs containing large amounts of yolk (insects, snails, fishes and newts) and for secretory organs containing high levels of proteinaceous material.

Insulin is present in chicken eggs and early chick embryos.

We showed that insulin appears in the chick embryo before beta-cells are recognizable, as well as in the egg constituents even before fertilization. Acid-ethanol extracts of 2- to 8-day-old embryos were gel-filtered on Sephadex G-50. The peak of immunoreactive insulin chromatographed in a position corresponding to that of authentic insulin. The immunoreactive insulin extracted from embryos was approximately 2 ng/g wet wt during early embryogenesis (days 2, 3, and 4), with a 2- to 3- fold increase by days 5 and 6, in conjunction with pancreatic development. The heads of the embryos contributed 22-23% of the total insulin on days 3 and 4, but only 5% by day 5. Based on its reactivity in a pork insulin RIA, chicken insulin RIA, and rat adipocyte bioassay, we concluded that the material is very similar to avian (chicken or turkey) rather than mammalian type insulin. Similar immunological and biological insulin-like activity [but at much lower concentrations (0.2-0.8 ng/ml)] were recovered from the gel-filtered acid-ethanol extracts of yolk and white of unfertilized and fertilized eggs. This study, which shows that insulin is present at a very early stage in ontogeny, extends observations that insulin is native to organisms that lack pancreatic islets, including flies, worms, and microbes.

Vitamin B12 bioavailability from egg yolk and egg white: relationship to binding proteins.

Egg yolk has been reported to inhibit B12 absorption less than egg white suggesting that different vitamin B12 binding proteins may be present in egg white and egg yolk. Using gel-exclusion chromatography we found that the mean MR for the B12 binding protein derived from egg yolk was 125,000, whereas that derived from egg white was 97,750. Heat treatment of the apoprotein differentially reduced the binding capacity of egg yolk and egg white in a time-dependent manner with the greatest decrease in binding capacity occurring with egg white. In contrast, heat treatment of the holoenzyme delineated the egg yolk as the more labile. These studies suggest that egg yolk and egg white contain distinct R binders which could explain the differential B12 absorption from egg yolk and egg white.

Purification of egg yolk immunoglobulins. A two-step procedure using hydrophobic interaction chromatography and gel filtration.

A two-step chromatographic procedure was developed for the isolation and purification of hen IgY antibodies from egg yolk. The antibodies were completely separated from vitellin and lipids by hydrophobic interaction chromatography followed by gel filtration. Almost no residual yolk proteins, no immunoglobulin aggregates, and no antibody fragments could be detected in the final extract. Moreover, the method described, guarantees the recovery of antibodies of undiminished activity. Although the final yield is somewhat lower than that obtained by an isolation method consisting of two precipitation steps with polyethylene glycol and alcohol respectively, the procedure described is particularly recommended when highly purified antibody preparations are needed.

Rapid method of extraction of antibodies from hen egg yolk.

Antibodies were raised in laying hens and isolated from the yolk of their eggs by precipitation with precooled (-20 degrees C) propane-2-ol and removing lipid material with propane-2-ol and acetone. The dried precipitate was extracted with phosphate buffer and shown to contain IgG antibodies and a small amount of additional protein. By Ouchterlony gel diffusion and rocket immunoelectrophoresis, the concentration and quality of specific antibodies in the extracts were comparable to those found in the serum of rabbits or to the IgG separated from yolk by other methods. The new isolation procedure is rapid, reliable and convenient.

Antibodies to rotaviruses in chickens' eggs: a potential source of antiviral immunoglobulins suitable for human consumption.

The prevalence of antibodies to human rotaviruses in commercially available eggs and egg products that are suitable for human consumption was investigated. The yolks of virtually all of the individual eggs and pasteurized pooled egg preparations contain antirotavirus antibodies detectable by means of enzyme immunoassay systems. Also, the eggs and egg preparations are capable of inhibiting the growth of two strains of rotaviruses in tissue culture. Chromatographic studies indicated that the antigen-binding activity is limited largely to the immunoglobulin fractions of the egg yolks. The antibody levels in eggs can be increased by the immunization of hens with purified rotavirus preparations, and the immunoglobulins isolated from the eggs of immunized hens can prevent the development of rotavirus gastroenteritis in experimentally infected animals. Egg preparations might serve as a practical source of antiviral antibodies suitable for consumption by infants and young children.

Comparison of the amino acid sequences of hen plasma-, yolk-, and white-riboflavin binding proteins.

The amino acid sequence of hen egg yolk-riboflavin binding protein (yolk-RBP) was determined by conventional methods. The sequence was identical with that of hen egg white-riboflavin binding protein except that their carboxyltermini were different, that of yolk-RBP lacked 11 or 13 amino acid residues, while hen plasma-RBP had the same C-terminal sequence as white-RBP. This indicated that the C-terminal 11 or 13 amino acid residues in plasma-RBP might be cleaved off during the incorporation from the blood into the oocyte or in the yolk fluid. Yolk-RBP had the same characteristics as white-RBP, such as N-terminal pyroglutamic acid, polymorphism in the amino acid sequence (Lys/Asn) at the fourteenth residue from the N-terminal end, carbohydrate chains attached to both Asn(36) and Asn(147) residues, and phosphate groups bound to some serine residues in the sequence of Ser(185) to Ser(197) as a cluster. These results led us to the conclusion that yolk- and white-RBPs are bio-synthesized from the same gene in the different organs (liver and oviduct). The carbohydrate composition of yolk-RBP was identical to that of plasma-RBP but different from that of white-RBP showing that the processing of the carbohydrate chains in the liver was different from that in the oviduct.

Thermodynamics of association of egg yolk riboflavin binding protein with 8-substituted riboflavins. Comparison with the egg white protein.

The association constants of hen egg yolk riboflavin binding protein with 8-substituted riboflavins were measured using a fluorometric titration method in the range of 10-40 degrees C, and thermodynamic parameters were calculated using ordinary methods. The flavins used were riboflavins whose 8-methyl group was substituted with HRN, RR'N, RO type groups, or halogen atoms. From a plot of delta H degrees against delta Su (unitary entropy change), the flavins were classified into four groups, i.e., HRN, RR'N, RO, and halogen type substituents. In each group, the linear free energy relationship, delta H degrees = u . delta Su+v (u and v were constants specific for each group), was found to be similar to that for egg white riboflavin binding protein, though with different specific constants for each egg protein. The relation between delta Su and the bulkiness (molecular volume) of 8-substituents suggested burying of the 8-substituents in a cavity of the protein similar to that of the egg white protein, but of smaller volume. The behavior of the halogen group was similar to that of the other three groups, and could be explained in terms of the bulkiness of the substituents rather than by assuming electric repulsion between halogen substituents and the binding site, which is in contrast with the case of egg white protein. A linear relation between v value and the wavelength of the visible absorption peak of flavins wa also found.

High performance liquid chromatographic analysis of long chain bases in intestinal glycolipids of adult and embryonic Japanese quails.

A new sensitive assay method for sphingoids, 4-D-hydroxysphinganine, sphingosine, and sphinganine, involving reversed phase HPLC was described. Chromatography of p-N-nitrophenylacetyl derivatives of the sphingoids showed sufficient resolution, and each peak showed a linear relationship between molar quantity and area response, from 10 pmol to 10 nmol. The method was applied to the analysis of long chain bases in intestinal glycolipids from embryonic and adult Japanese quails. At the embryonic stage of 12 days' incubation, 4-D-hydroxysphinganine accounted for about 40% of the long chain bases of glycolipids, a concentration comparable to that in adult tissue. Egg yolk, which is an exclusive exogenous nutrient mixture supplied by the mother, contained only a few percent of 4-D-hydroxysphinganine as a glycolipid component. While, [3H]palmitic acid administered to embryos was incorporated into 4-D-hydroxysphinganine as well as sphingosine and sphinganine. These results suggest that sphingoids of intestinal glycolipids including 4-D-hydroxysphinganine are synthesized in the tissue, excluding the possibility of their exogenous origin in the embryonic tissue.

Egg globulins in rapid virus diagnosis.

Egg-derived antibodies specific for a range of human viruses are now available commercially. These globulins are prepared by inoculating chickens with the relevant virus then harvesting antibodies from egg yolks. Reagents for influenza A and B, parainfluenza 1 and 3, adenovirus group antigen and rotavirus were tested. The successful use of these reagents on tissue culture and clinical material is described as well as their quality assessment. The advantages of this type of antibody are discussed.