Two distinct yolk lipoprotein complexes from Caenorhabditis elegans

The four yolk polypeptides of the nematode Caenorhabditis elegans are found in two types of lipoprotein particle: 12 S particles with Mr estimated at 450,000 and 8 S particles with Mr estimated at 250,000. Both types of particle contain approximately 8% phospholipids, 3% triglycerides, and 3% other lipids by mass. All four C. elegans yolk polypeptides can be found in either 12 or 8 S particles, depending upon the conditions of isolation. While the properties of the 12 and 8 S lipoprotein particles are consistent with a dimermonomer relationship, the asymmetric distribution of the yolk polypeptides between 12 and 8 S fractions suggests that at least two different oligomeric lipoprotein complexes are present in C. elegans embryos. In order to clarify the subunit composition of the C. elegans yolk lipoproteins, the patterns of polypeptides retained in immunoaffinity binding procedures by immunoglobulins of different antigenic specificities have been compared. When immunoaffinity binding is performed in the absence of sodium dodecyl sulfate, three C. elegans yolk proteins (yp170A, yp115, and yp88) are retained together by polyclonal immunoglobulins directed against either yp115 or yp88. A monoclonal immunoglobulin also retains these three proteins together. In contrast, a second monoclonal immunoglobulin retains only the fourth yolk protein (yp170B). Aggregate species, evidently reflecting the spontaneous formation of interchain disulfide bonds, indicate that yp170A and yp88 are physically associated, whereas yp170B self-associates in dimers. It is concluded that there are two distinct lipoprotein complexes in C. elegans: the A complex, which consists of yp170A, yp115, and yp88 and is essentially heterodimeric and the B dimer, a simple dimer of yp170B.     

Effects of 2,3-diketo-L-gulonic acid on the oxidation of yolk lipoprotein

2,3-Diketo-L-gulonic acid (DKG) is an important intermediate product of oxidative degradation of L-ascorbic acid (AsA) in both biological and food systems, but the physiological function of DKG is still unclear. In this study, it was found that DKG had a strong antioxidative effect on copper-dependent oxidative modification of yolk lipoprotein (YLP), on the basis of both the decreased electrophoretic mobility and longer lag time of conjugated diene formation in a concentration-dependent manner. DKG is known to be very unstable and easily converts into two delta-lactones of DKG, the 3,4-enediol form of DKG delta-lactone (3,4-DKGL) and 2,3-enediol form of DKG delta-lactone (2,3-DKGL) depending on both pH and temperature. 3,4-DKGL was thought to be the first degradation product of DKG and could play an antioxidative role in the oxidation of lipoproteins induced by copper ion or peroxyl radicals in neutral aqueous solution.     

Characteristics of the structure of plasma lipoprotein crystals

Lipoproteids of human blood plasma in the norm and in pathology are studied by means of microscopy in polarized light. It is shown that lipoproteids in solid state have structure characteristics. Possible reasons of the observed structure characteristics of lipoproteid crystals are discussed.     

Lipoprotein crystals in the yolk platelet of a teleost,Pelvicachromis pulcher (Cichlidae)

Summary Yolk-platelet crystals in the oocytes of the teleost Pelvicachromis pulcher (Cichlidae) were shown, using electron diffraction and tilting of thinsectioned specimens, to possess an orthorhombic lattice with unit-cell sides a=8.3 nm, b=16.6 nm and c=18.0 nm. They thus closely resemble the crystals known for a newt (Triturus sp.) and a frog (Xenopus laevis).Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg. The author wishes to thank Dr. R. Riehl, Heidelberg, for taxonomic advice     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

Preparation of lipid-free protein extracts of egg yolk

1-Butanol extraction of chicken egg yolk homogenates containing 1 m NaCl yields lipid-free aqueous solutions of egg yolk proteins. These solutions, after dialysis, can be applied to a variety of chromatographic media without clogging. Although some proteins are denatured by this procedure, most of the water-soluble proteins remain in solution, including biotin-binding protein and riboflavin-binding protein.     

Apolipoproteins and lipoprotein families in chicken embryos and egg yolk.

1. Turkey anti-LP-A and anti-LP-B cross react strongly with whole adult chicken serum. 2. Similar reactions occur with whole chicken embryo sera (10-21 days of incubation) and egg yolk fluid. 3. Ultracentrifugation of sera from 14-day old chicken embryos, after reaction with anti-LP-B, reveal complete precipitation of the VLDL and LDL of embryo serum (indicating that adult-type LP-B is the major apolipoprotein of these density classes). 4. All of the chicken embryo adult-type apo-LP-A is in the HDL fraction. 5. However, egg yolk and hen serum VLDL contain apo-LP-A as well as apo-LP-B.     

The receptor for yolk lipoprotein deposition in the chicken oocyte.

The final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically synthesized yolk precursor proteins from the plasma. The two major yolk-forming components, very low density lipoprotein (VLDL) and vitellogenin (VTG), have been shown to interact with a 95-kDa protein present in detergent extracts of ovarian membranes; this protein is absent in hens of a mutant nonlaying chicken strain (Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398). Here, we have purified the 95-kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor-mediated endocytosis by ultrastructural immunolocalization, structural, and functional studies. The receptor was visualized exclusively in the oocyte proper and was absent from somatic cells, in agreement with the previously reported expression of two different lipoprotein receptors in somatic cells and oocytes, respectively, of laying hens (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Amino acid sequences of tryptic fragments of the oocyte receptor were obtained, and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains. In particular, the chicken receptor's internalization sequence, Phe-Asp-Asn-Pro-Val-Tyr, is identical with that in low density lipoprotein receptors from mammals as well as Xenopus laevis. The ligand-binding properties, specificity, and kinetic parameters of the oocyte receptor were characterized in filtration assays employing pure ligands and receptor. In conjunction with ligand-blotting experiments following limited protease digestion of the receptor, the binding assay data suggest that VTG recognizes a substructure of the VLDL-binding site. These studies establish that a cell-specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays a key role in control of oocyte growth.     

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 g/ml (4 g 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.