Yolk Lipoproteins Research Articles

A novel isolation method for hen egg yolk antibody, "IgY".

A method for isolation of egg yolk immunoglobulin, IgG, a livetin protein, was investigated. Several natural gums (carrageenan and xanthan gum) were found to be effective for removal of yolk lipoprotein as a precipitate. The effect was pronounced with lambda-carrageenan and the lipid content in the supernatant after removal of the resulting precipitate was less than 0.4% of that of egg yolk. IgY remained in this supernatant, with a yield of 86%, and was isolated by chromatography on a column of DEAE-Sephacel followed by salting-out with sodium sulfate. IgY thus isolated was almost pure (98%) and the yield was 70 to 100 mg per egg.

Screening of immunoglobulin production stimulating factor (IPSF) in foodstuffs using human-human hybridoma HB4C5 cells.

We screened the immunoglobulin production stimulating factor (IPSF) in foodstuffs, using human-human hybridoma HB4C5 cells cultured in serum-free 1TES-ERDF medium, and found that egg yolk lipoprotein (YLP), lactoferrin, Block Ace, and casein had IPSF activity. The maximum IPSF activity was obtained at concentrations over 100^g/ml in YLP, 10μg/ml in lactoferrin, and 25 μg/ml in Block Ace and casein. These IPSFs stimulated the IgM production of human-human and mouse-mouse hybridomas, but their effect on IgG producers was very small. This suggests that IgG production of hybridomas is regulated differently from their IgM production.

Role of lipophorin in lipid transport to the insect egg.

Lipid accounts for 40% of the dry weight of a mature Manduca sexta egg. Less than 1% of the total egg lipid is derived from de novo synthesis by the follicles. The remaining egg lipid originates in the fat body and is transported to the ovary by lipoproteins. Vitellogenin, the major egg yolk lipoprotein, accounts for 5% of the total egg lipid. The remaining 95% lipid is attributable to the hemolymph lipophorins, adult high density lipophorin (HDLp-A) and low density lipophorin (LDLp). When HDLp-A that is dual labeled with 3H in the diacylglycerol fraction and 35S in the protein moiety is incubated with follicles in vitro, the ratio of 3H:35S in the incubation medium does not vary and is similar to the ratio of the labels that are associated with the follicles. In an accompanying paper (Kawooya, J. K., Osir, E. O., and Law, J. H. (1988) J. Biol. Chem. 263, 8740-8747), we show that HDLp-A is sequestered by the follicles without subsequent hydrolysis of its apoproteins. These results, together with those presented in this paper, support our conclusion that HDLp-A is not recycled back into the hemolymph after it is internalized by the follicles and, therefore, does not function as a reusable lipid shuttle between the fat body and the ovary. When follicles are incubated with dual labeled LDLp, the diacylglycerol component of the particle is internalized by the follicles without concomitant endocytosis of its associated apoproteins. This LDLp particle is the major vehicle by which lipid is delivered to the ovary.

Egg yolk lipoprotein, a new supplement for the growth of mammalian cells in serum-free medium

Egg yolk lipoprotein promoted growth of a wide variety of mammalian cell lines, including plasma-cytomas and epithelial cell lines, in serum-free medium. The lipoprotein was active for cell growth when used with insulin, transferrin, ethanolamine and selenite. The most active lipoprotein fraction (YLP-pI7.5) was purified to give a single peak by chromatofocusing and gel filtration, and was homogeneous on a 0.35% agarose gel electrophoretogram. The lipoprotein was characterised as a very low density lipoprotein with a protein content of only 1.3%. This lipoprotein had an optimal concentration of 300 μg/ml (4 μg protein/ml). It was easily separable from proteinous molecules secreted into the serum-free medium by the cells, since it floated on the surface of the medium after addition of ammonium sulfate, to precipitate protein, and centrifugation. An associated structure of lipid and protein seemed to be still necessary for the lipoprotein to exhibit a growth promoting activity.

Regulation of expression of Xenopus vitellogenin genes.

The assembly of the developing egg is a fascinating example of division of labor among oocytes and other tissues, with the coordination of activities in different tissues being achieved by hormones. Among nonoocyte tissues are (1) the follicle cells, which, under the influence of follicle-stimulating hormone (FSH), synthesize and release the sex steroids estrogen and progesterone; (2) the oviduct, which produces egg white proteins, coat proteins, or egg-sustaining fluids and whose activity is regulated by both steroid hormones; and (3) the liver, which, under the sole control of estrogen, provides the developing egg with yolk proteins, cholesterol, phospholipids, and lipoproteins. The participation of the oviduct and liver is particularly pronounced in nonmammalian species with free-living embryos, as in birds and amphibia.

Physicochemical study of rock crab lipoproteins

Physicochemical studies have been carried out on the hemolymph and egg lipoproteins of the rock crab ( Cancer antennarius). Analytical ultracentrifugal analyses of vitellogenic female HDL 3 revealed the presence of two types of lipoproteins. The first with a sedimentation rate of 5.35 S was comparable to lipoproteins in male and non-vitellogenic female hemolymph. The second with a sedimentation rate of 10.74 S was comparable to the major lipoprotein of egg yolk. A similar comparison could be made following electrophoretic analyses in native polyacrylamide gels. Electrophoresis in SDS-polyacrylamide gels revealed three major apolipoproteins common to egg and vitellogenic HDL 3. A fourth apolipoprotein was found in both male and female HDL 3. In contrast to mammalian HDL, none of these crustacean apolipoproteins had a molecular weight less than 82000. One of these apolipoproteins appears to be comparable physicochemically to the enteric form of apolipoprotein B in mammals.

Preparation of single crystals of a yolk lipoprotein

Single crystals of a yolk lipoprotein from two species of lamprey oocytes have been prepared and shown to belong to the monoclinic space group C2. The unit cell dimensions combined with molecular weight determinations support the idea that the lipoprotein is a symmetrical dimer centered on a crystallographic 2-fold symmetry axis. The lipid content is estimated to be about 15% (w/w) and the crystals are suitable for structural studies by single crystal X-ray analysis. The X-ray results are correlated with those obtained by electron diffraction.

Immunochemical investigation of the interaction of egg-yolk lipoproteins with bovine spermatozoa.

Antiserum was raised in rabbits against an egg-yolk lipoprotein fraction previously shown to have cryoprotective properties. It was used in conjunction with alkaline phosphatase-conjugated goat anti-rabbit IgG to demonstrate lipoprotein interaction with bovine spermatozoa. The lipoprotein bound firmly to spermatozoa and was not removed by extensive washing, suggesting that the lipoprotein had become irreversibly associated with the sperm membranes.

Studies on the Apoproteins of the Major Lipoprotein of the Yolk of Hen's Eggs. V. Protein Sulfhydryl Groups: Accessibility in the Lipoprotein and the Effect of Temperature

Sulfhydryl groups in the apoproteins of the major lipoprotein (density 0.95 g/ml) of hen's egg yolk have been studied by colorimetry, analysis for carboxymethylcysteine, and labelling with [14C]iodo-compounds and their distribution amongst the individual apoproteins (i.e. apovitellenins I-VI) has been determined. In the intact lipoprotein, reaction of sulfhydryl groups with aqueous reagents was slower than in the apoprotein mixture and there were large differences amongst different reagents in the extent of reaction. If the lipoprotein was heated for a short time in 1 mM EDTA above about 70 degrees C and then cooled, more sulfhydryl groups were accessible although the total number did not change. This increase in accessibility was accompanied by the disruption of the lipoprotein structure and an increase in the viscosity of its solutions. Furthermore, the isolated apoprotein mixture was less soluble in urea solutions and formed a gel, probably as a result of cross-linking by inter-protein disulfides. There was also a small change in the distribution of sulfhydryl groups so it is likely that sulfhydryl-disulfide interchanges occurred on heating. It is suggested that such interchanges help determine the physical properties of heated yolk and that in the intact lipoprotein sulfhydryl groups are concealed to prevent sulfhydryl-disulfide interchanges in the egg.

Adsorption of pancreatic (pro)phospholipase A2 to various physiological substrates.

The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.

The influence of egg yolk lipoproteins on the rheology and stability of O/W emulsions and mayonnaise

Several variables influence the time dependent reduction of interfacial tension by egg yolk lipoproteins at a groundnut oil-water interface. These are pH, NaCl, methanol and temperature, and they affect the degree of micelle breakdown and subsequent molecular unfolding and rearrangement following adsorption. Diffusion to the interface is a rate controlling factor when the aqueous phase contains a very low concentration of egg yolk, but only in the early stages of adsorption. Otherwise, penetration of the interface by low density lipoproteins, breakdown of micelles and configurational changes predominate.

The influence of egg yolk lipoproteins on the rheology and stability of O/W emulsions and mayonnaise

The influence of egg yolk lipoproteins on the rheology and stability of O/W emulsions and mayonnaise

Hydrophobic chromatography of proteins in urea solutions. The separation of apoproteins from a lipoprotein of avian egg yolk

A method is described for the chromatographic separation of mixtures of egg-yolk proteins of low solubility by using a hydrophobic column (phenyl-Sepharose) and eluting with increasing concentrations of aqueous urea at low pH. The resolving power of the method was established by tests on proteins and protein fragments of known sequence. The theoretical basis for the method remains, however, unclear. Factors such as the aggregation of the protein often appeared to be more important than its hydrophobicity in determining the urea concentration needed for elution. The method was applied to the mixture of apoproteins from the low-density lipoprotein (density about 0.95 g/ml) of avian egg yolk. For the previously studied apoproteins from egg yolk of the hen (Gallus domesticus), hydrophobic chromatography provided a new and convenient method for isolating the main apoproteins (hen apovitellenins I-VI). For the hitherto unexplored apoproteins from egg yolk of the duck (Anas platyrhynchos) the method has now been used to isolate three new proteins, two of which were not readily separated by methods based on molecular size. The elution pattern obtained with duck egg-yolk apoproteins is not the same as that of the hen egg-yolk apoproteins, although we suggest a relationship for the three new apoproteins based on their amino acid compositions and other properties. Possible roles for the apoproteins in avian egg yolk are described.

Female sex pheromone in the skin and circulation of a garter snake.

Serums and extracts of tissues from the female garter snake (Thamnophis sirtalis parietalis) each act as a pheromone and elicit male courtship behavior when applied to the back of another male. Since pheromonal activity is present in yolk and liver tissue of untreated females and can be induced with estrogen treatment in the serums and livers of males, the pheromone may be associated with the circulating yolk lipoprotein, vitellogenin.

A spectrofluorometric investigation, using 1-anilino-naphthalene-8-sulphonate, of the interaction between washed bovine spermatozoa and seminal plasma or egg-yolk lipoprotein.

The dye 1-anilino-naphthalene-8-sulphonate (ANS) bound at 94.5 +/- 2.7 pmol per 10(6) spermatozoa (KD = 1.83 +/- 0.13 X 10(-5) M) and 7.30 +/- 0.17 pmol per mg seminal plasma protein (KD = 2.19 +/- 0.06 X 10(-5) M). Equilibration of excess ANS with a mixture of washed spermatozoa and seminal plasma resulted in a significant increase in fluorescence over that calculated by summation of the individual levels (P less than 0.05). A less pronounced but significant increase was seen when an egg-yolk lipoprotein, previously shown to have cryoprotective properties, was similarly added to washed spermatozoa (P less than 0.05). This increase in fluorescence was not reduced by washing spermatozoa--lipoprotein mixtures, suggesting that an interaction between the lipoprotein and the cell membrane had occurred.

The roles of lipid and protein in the protection of ram spermatozoa at 5 degrees C by egg-yolk lipoprotein.

Both the low-density lipoprotein fraction of egg yolk (LDF) and sonicated lecithin liposomes provided an equal measure of protection for ram spermatozoa during cold shock, but LDF was superior to lecithin during cold storage. The protective activity of LDF during storage at 5 degrees C was not altered by a subfractionation procedure which did not alter the molecular organization. Removal of the protein from the surface of LDF particles gave preparations with altered lipid:protein ratios. Neither the low-lipid fraction nor bovine plasma albumin protected spermatozoa but the high-lipid fraction was as protective as LDF. Survival of spermatozoa decreased as the lipid:protein ratio fell below 1.67 compared with a ratio of 4.76 for LDF. The absolute lipid content was more important than the ratio except at low ratios. Lipid-depleted preparations bound more effectively to the plasma membrane than did LDF whereas the lipid-enhanced preparation showed little binding capability. It is concluded that the phospholipid of LDF provides the protection to the sperm cell membrane. The protein of LDF serves to solubilize the lipid and bind it to the cell membrane. The importance of the role of the protein during cold storage is discussed.

A symmetric lipovitellin-phosvitin dimer in cyclostome yolk platelet crystals: Structural and biochemical observations

Lipoprotein crystals of hagfish yolk platelets ( Myxine glutinosa L.) are, by electron diffraction of embedded specimens, monoclinic ( a > 19.8 nm, b > 8.9 nm, c > 9.0 nm, β ~ 105 °; space group C2: 2 dimers per cell). Using sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the four major protein bands in Myxine (molecular weights 30,000; 44,000: 80,000 and 130,000) correspond, with small differences, to similar bands of Xenopus yolk lipoproteins. The symmetric lipovitellin-phosvitin dimer in cyclostomes is unique and probably reflects the lack of diversification of homologous vertebrate protein species.

Cholesterol and phospholipids of bovine spermatozoa. I. Selection of a PIPES-buffered diluent for evaluating the effect of egg yolk lipoproteins on sperm cholesterol and phospholipids.

Cholesterol and phospholipids of bovine spermatozoa. I. Selection of a PIPES-buffered diluent for evaluating the effect of egg yolk lipoproteins on sperm cholesterol and phospholipids.

Microcomplement fixation and particle size of chicken lipoproteins

Conformational changes of the lipoproteins of chicken serum and egg yolk were studied with the use of microcomplement fixation. Egg yolk very low density lipoproteins and serum total low density lipoproteins were separated by 2% agarose gel chromatography into four and five fractions, respectively, with different average particle diameters. A correlation between percent complement fixed at equivalence and particle diameter was noted. Thus, smaller particles fixed less complement. The shortening of the radius of curvature could alter the steric fit of apolipoprotein with antibody and, as a consequence, reduce complement fixation. The patterns obtained after delipidation of very low density lipoproteins indicate a general distortion of apolipoprotein conformation supporting the view that lipids are essential for maintaining the conformation of these apolipoproteins. On the other hand, the general conformation of the high density apolipoproteins does not appear to depend on lipid content. It has been reported that lipids restrain the free movement of these apolipoproteins. After delipidation, the greater degree of freedom allowed the apolipoprotein may increase the strength of the antigen-antibody union and therefore an increase in complement fixation is observed. These studies demonstrate the applicability of quantitative microcomplement fixation to the eludication of lipoprotein structure and function.

Lipoprotein lipase: some effects of activator proteins.

This paper considers how apolipoprotein CII from human plasma lipoproteins and T1 and T2 proteins from egg yolk lipoproteins stimulate the activity of lipoprotein lipase. These activator proteins stabilized the enzyme much more effectively than a thousandfold higher concentration of albumin did, indicating a direct interaction with the enzyme. The effects of the activators were seen also at 1 M NaCl. Thus, forces other than electrostatic are implicated. Centrifugation experiments showed that 125I-labeled lipase bound equally well to the emulsion droplets in the absence of activator protein as in its presence. This was true even under conditions when the activator caused a severalfold increase in the rate of hydrolysis. Thus, the activator makes enzyme at the interface more effective in hydrolysis. By optimizing the conditions it was possible to obtain almost as high rates of triglyceride hydrolysis in the absence as in the presence of activator. Thus, the main effect of the activator protein is probably not on a rate-limiting chemical step. Under most conditions, the rate of hydrolysis was much below optimal and activator increased it. This was always the case with phosphatidyl-choline/triglyceride emulsions, where the activator enhanced hydrolysis of both lipids. Other experiments showed that the activator enhanced triglyceride hydrolysis in the absence of phospholipids and phospholipid hydrolysis in the absence of triglycerides. It is suggested that interaction with activator orientates the enzyme and/or the lipid substrate for effective hydrolysis at the surface of lipoproteins/model substrates.