Apolipoprotein specificity of the chicken oocyte receptor for low and very low density lipoproteins: lack of recognition of apolipoprotein VLDL-II

At onset of egg-laying in the chicken, plasma levels of apolipoprotein VLDL-II (apoII) increase dramatically, suggesting a function of apoII in yolk deposition of triglyceride-rich lipoproteins. Thus, the possibility that this female-specific homodimeric protein (Mr of subunit, 9500) is recognized by the oocyte receptor for low and very low density lipoproteins was investigated. ApoII was purified from very low density lipoproteins by a novel, rapid procedure and reconstituted with egg phosphatidylcholine (PC) by detergent-dialysis. The resulting discoidal apoII/PC lipoprotein particles contained 3 mg of apoII per mg of PC and had a buoyant density of 1.062 g/ml. The ability of apoII/PC, as well as of physiological particles containing apoII but devoid of apolipoprotein B (apoB), namely high density lipoproteins (HDL) from laying hens, to interact with the oocyte receptor was tested. Both of these ligands failed to show saturable high affinity binding, in contrast to the apoB-containing ligands, low and very low density lipoproteins. Furthermore, neither laying-hen HDL which contain apoII and apoA-I nor apoII/PC were able to displace receptor-bound apoB-containing lipoproteins, as shown in competitive binding assays as well as by ligand blotting. Thus, we conclude that apoB, but not apoII, participates in binding and uptake of very low density lipoproteins via receptor-mediated endocytosis by growing chicken oocytes.     

The isolation and partial characterization of the serum lipoproteins and apolipoproteins of the rainbow trout.

1. VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins were isolated from the serum of trout (Salmo gairdneri Richardson). 2. Each lipoprotein class resembled that of the human in immunological reactivity, electrophoretic behaviour and appearance in the electron microscope. Trout LD lipoprotein, however, was of greater density than human LD lipoprotein. 3. The trout lipoproteins have lipid compositions which are similar to those of the corresponding human components, except for their high contents of long-chain unsaturated fatty acids. 4. HD and LD lipoproteins were immunologically non-identical, whereas LD lipoproteins possessed antigenic determinants in common with VLD lipoproteins. 5. VLD and HD lipoproteins each contained at least seven different apoproteins, whereas LD liprotein was composed largely of a single apoprotein which resembled human apolipoprotein B. 6. At least one, and possibly three, apoprotein of trout HD lipoprotein showed features which resemble human apoprotein A-1.7. The broad similarity between the trout and human lipoprotein systems suggests that both arose from common ancestral genes early in evolutionary history.     

High-affinity binding of lower-density lipoproteins to chicken oocyte membranes.

Oocyte membrane fragments bind specifically radioiodinated VLD lipoprotein (very-low density lipoprotein) and LD lipoprotein (low-density lipoprotein). Competitive binding assays showed 2-3 times more VLD lipoprotein than LD lipoprotein bound at 4 degrees C. Equilibrium-binding data revealed the presence of one class of non-interacting sites for VLD lipoprotein (kD 12 microgram/ml) and co-operative binding for LD lipoprotein. The binding of VLD lipoprotein showed a distinct pH maximum at 5.3, whereas an indistinct maximum at about pH 7.3 was observed for LD lipoprotein. Unlabelled VLD lipoprotein did compete with 125I-labelled LD lipoprotein binding, but unlabelled LD lipoprotein did not compete with 125I-labelled VLD lipoprotein binding. VLD lipoprotein binding was inhibited by HD lipoprotein (high-density lipoprotein), but not by lysozyme, collagen, poly-L-lysine or poly-L-arginine; LD lipoprotein binding was inhibited by lysozyme and collagen, but not by HD lipoprotein. On the basis of these studies, we suggest that: (1) VLD lipoprotein and LD lipoprotein enter the oocytes by a receptor-mediated transport mechanism; (2) the receptors for VLD lipoprotein and LD lipoprotein are distinct; and (3) the binding of LD lipoprotein to chicken oocyte membranes differs from that to other cell types.     

The distribution and partial characterization of the serum apolipoproteins in the guinea pig.

1. Very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins were isolated by sequential ultracentrifugation from the serum of male guinea pigs fed on a diet containing 3--4% fat. The apoproteins of these lipoproteins (apo-VLD, apo-LD and apo-HD lipoproteins) were studied after delipidation with organic solvents or extraction with tetramethylurea. 2. The major apolipoprotein of LD lipoprotein isolated by gel filtration was found to closely resemble apolipoprotein B of human serum in its chemical and physical properties. Electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that this apoprotein consisted of a number of polypeptides. 3. Tetramethylurea precipitated an apoprotein from guinea-pig serum lipoproteins that is probably the apolipoprotein B-like component. This apoprotein accounted for about 80% of the apo-LD lipoprotein, about 55% of the apo-VLD lipoprotein and about 50% of the apo-HD lipoprotein. 4. The distribution of apolipoproteins soluble in tetramethylurea was determined by densitometric scanning of stained polyacrylamide disc gels. 5. A glycine-rich component of high electrophoretic mobility (band I) and a triplet of soluble apolipoproteins (bands II-IV) were present in both VLD and LD lipoprotein classes. These components constituted a higher proportion of the tetramethylurea-soluble apoproteins of VLD lipoprotein (60--80%) than of LD lipoprotein (40--55%). 6. Small amounts (10--15%) of a component of intermediate mobility, which contained traces of half-cystine, were also present in both VLD and LD lipoproteins. 7. A group of soluble components of basic character (bands VI-X), present as minor components of VLD lipoprotein (10--20%), constituted a major proportion (30--45%) of the soluble apoproteins of LD lipoprotein. Two of these apoproteins were rich in lysine, and two of lower electrophoretic mobility were rich in arginine. 8. The pattern of tetramethylurea-soluble apoproteins in HD lipoprotein was distinguished by the presence of two polypeptides of low electrophoretic mobility as its predominant components. One of these components, band VI, resembled the A-I apolipoprotein of man in both its amino acid profile and in its electrophoretic mobility. The second major component, band VI-B, was rich in lysine and resembled the C-I apolipoprotein of man in amino acid composition. 9. The soluble components of bands I and IX were analogous in physicochemical properties to the R-X1 and R-X2 (high-arginine polypeptide) peptides of human serum lipoproteins respectively.     

Lipoprotein particles from the Golgi apparatus of guinea-pig liver

1. A cell fraction has been isolated from guinea-pig liver and shown to be rich in Golgi apparatus by electron microscopy. The activity of UDP-d-galactose-N-acetylglucosamine galactosyltransferase was over 100-fold greater in this cell fraction than in the liver homogenate. These data support the conclusion that the fraction was enriched in Golgi apparatus. 2. The Golgi cisternae and secretory vesicles contained electron-dense particles of 10-80nm diameter. Disruption of the Golgi apparatus cell fraction released these particles, which were separated into VLD (very-low-density) and LD (low-density) species on the basis of their density. 3. The Golgi VLD particles possessed morphological, flotational, chemical and immunochemical properties which closely resembled those of the serum VLD lipoproteins from the same animals. 4. The Golgi LD particles were rich in phospholipid, containing 48.1% by weight. The chemical composition of these particles was quite distinct from that of the serum LD lipoproteins, but did, however, show some similarity to that of the serum VLD lipoproteins. A marked resemblance was noted in the chemical characteristics of the Golgi LD and VLD particles (with the exception of triglyceride content). In addition, these two species of Golgi particles possessed the same antigenic determinant. 5. The results suggest that the Golgi VLD particles are the precursors of the serum VLD lipoproteins. On the basis of similarities in gross chemical composition and in the antigenic determinant of the Golgi LD and VLD particles, we conclude that the LD particles are probably the precursors of the VLD particles. In view of the marked differences in gross chemical composition of the Golgi LD particles and serum LD lipoproteins, it appears unlikely that the LD particles are directly secreted into the serum pool.     

The metabolic conversion of very-low-density lipoprotein into low-density lipoprotein by the extrahepatic tissues of the rat.

1. The work reported was designed to provide quantitative information about the capacity of the extrahepatic tissues of the rat to degrade injected VLD lipoproteins (very-low-density lipoproteins, d less than 1.006) to LD lipoproteins (low-density lipoproteins, d 1.006--1.063) and to study the fate of the different VLD-lipoprotein apoproteins during the degradative process. 2. Rat liver VLD lipoproteins, radioactively labelled in their protein moieties, were produced by the perfusion of the organ and were either injected into the circulation of the supradiaphragmatic rats or incubated in rat plasma at 37 degrees C. At a time (75 min) when approx. 90% of the triacylglycerol of the VLD lipoproteins had been hydrolysed the supradiaphragmatic rats were bled and VLD lipoproteins, LD lipoproteins and HD lipoproteins (high-density lipoproteins, d 1.063--1.21) were separated from their plasma and from the plasma incubated in vitro. The apoproteins of each of the lipoprotein classes were resolved by gel-filtration chromatography into three main fractions, designated peaks I, II and III. 3. Incubation of the liver VLD lipoproteins in plasma in vitro led to the transfer of about 30% of the total protein radioactivity to the HD lipoproteins. The transfer mainly involved the peak-II (arginine-rich and/or apo A-I) and peak-III (apo C) proteins. There was also a small transfer of radioactivity (about 5% of the total) to the LD lipoproteins. 4. Injection of the liver VLD lipoproteins into the circulation of the supradiaphragmatic rat resulted in the transfer of about 15% of the total VLD-lipoprotein radioactivity to the LD lipoproteins. The transfer involved mainly the peak-I (apo B) proteins and accounted for about 20% of the total apo B protein radioactivity of the injected VLD lipoproteins. When the endogenous plasma VLD lipoprotein was taken into account the transfer of apo B protein was about 35%. 5. The transfer of peak-II protein radioactivity from the VLD to the HD lipoproteins was greater in the plasma of the supradiaphragmatic rat than in the incubated plasma suggesting that there was a net transfer of peak-II apoproteins during the VLD lipoprotein degradation. The transfer of peak-III protein radioactivity was not greater in the plasma of the supradiaphragmatic rat, but there was a loss of this radioactivity from the circulation.     

Oocytes from the mutant restricted ovulator hen lack receptor for very low density lipoprotein

Hens of the "Restricted Ovulator" (R/O) chicken strain are characterized by the absence of egg-laying and concomitant severe hyperlipidemia due to a single gene defect (Ho, K. J., Lawrence, W. D., Lewis, L. A., Liu, L. B., and Taylor, C. B. (1974) Arch. Pathol. 98, 161-172). However, the underlying biochemical defect has not been identified. Previous studies on receptor-mediated growth of chicken oocytes have led to the characterization of a 95-kDa oocyte plasma membrane receptor that binds very low density lipoproteins (VLDL) (George, R., Barber, D. L., and Schneider, W. J. (1987) J. Biol. Chem. 262, 16838-16847). The current experiments demonstrate the absence of this receptor from R/O oocytes. Ligand binding experiments showed that ovarian membranes from mutant hens failed to display high affinity, saturable, and specific binding of 125I-VLDL. Ligand blotting with 125I-VLDL and Western blotting with polyclonal anti-receptor antibodies visualized the 95-kDa receptor in normal oocytes, but R/O ovarian membranes were devoid of any cross-reactive protein. Finally, plasma clearance of intravenously injected 125I-VLDL was dramatically impaired in R/O in comparison to normal hens, with a concomitant decrease in the radioactivity accumulating in R/O oocytes. These data strongly suggest that the absence of the 95-kDa receptor for VLDL from oocytes is responsible for the R/O phenotype, and that the receptor not only binds VLDL, but also mediates its uptake. This animal model provides a powerful tool for investigations of receptor-mediated growth of chicken oocytes and for the elucidation of regulatory mechanisms in lipid and lipoprotein metabolism of laying hens.     

The effect of a lipid-rich diet on the properties and composition of lipoprotein particles from the Golgi apparatus of guinea-pig liver

1. A cell fraction rich in Golgi apparatus was isolated from the livers of guinea pigs fed on a lipid-rich diet (1.6% cholesterol, 15% corn oil). 2. The Golgi cisternae and secretory vesicles contained electron-dense particles which were tentatively identified as VLD (very-low-density) and LD (low-density) lipoproteins. Particles of moderate electron density, 150–500nm in diameter, were seen associated with membranous elements of the Golgi-apparatus cell fraction. Disruption of this cell fraction permitted the release of these three species of particles, which were separated into particulate lipid, and VLD and LD lipoproteins. 3. The large particles of moderate electron density, isolated as particulate lipid, were distinct from both species of Golgi particles in their chemical composition and in possessing an immunochemically unreactive apolipoprotein(s). Morphological observations suggest that the particulate lipid arose from cytoplasmic lipid droplets which were present as contaminants of the Golgi-rich fraction. 4. The chemical and immunochemical results are consistent with the suggestion that the Golgi LD particles are precursors of the VLD particles, into which they may be transformed by the addition of both triglyceride and cholesteryl ester. The present results provide further support for the proposal that the Golgi VLD particles are precursors of the serum VLD lipoproteins in the guinea pig. 5. Hepatic Golgi VLD particles isolated from guinea pigs fed on the lipid-rich diet contained significantly higher molar amounts (relative to protein) of both cholesteryl ester and triglyceride than similar particles from animals fed on a normal diet. These results suggest that the type of Golgi VLD particle produced from the LD particle is a direct consequence of the amount and composition of the dietary lipid. 6. Hepatic Golgi LD particles isolated from guinea pigs fed on different diets were similar in chemical composition and contained approx. 50% by weight of phospholipid. We conclude that the Golgi LD particle is normally present in the Golgi-apparatus cell fraction from guinea-pig liver, and may represent the end product of lipoprotein biosynthesis in the smooth endoplasmic reticulum. 7. The serum LD lipoproteins and Golgi LD particles were quite distinct in chemical composition. However, these two lipoprotein species were immunochemically identical and exhibited a similar range of flotation rate. It appears unlikely that the Golgi LD particles are secreted as the precursors of the serum LD lipoproteins.     

Exogenous cholesterol transport in rabbit plasma lipoproteins

1. The appearance of exogenous cholesterol in free cholesterol and ester cholesterol of plasma chylomicra, very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins was studied in unanaesthetized rabbits after ingestion of a meal containing 5% fat and 0.08% [³H]cholesterol. 2. The specific radioactivity of ester cholesterol of VLD lipoproteins reached the highest value of any lipoprotein fraction and for each lipoprotein it increased at a faster rate and reached a higher maximum than that of free cholesterol; the maximum in VLD lipoproteins occurred later than in chylomicra. 3. The pattern of appearance of exogenous cholesterol in chylomicra and VLD lipoproteins of plasma was similar to the pattern previously observed in lymph. The specific radioactivity of ester cholesterol in plasma VLD lipoproteins was higher than that in chylomicra in spite of a larger pool size and dilution of cholesteryl esters from VLD lipoproteins produced by the liver. These results support the concept that during absorption the major portion of exogenous cholesterol is transported in VLD lipoproteins as ester cholesterol. 4. The specific radioactivity of ester cholesterol of chylomicra and VLD lipoproteins increased at a faster rate than that of LD and HD lipoproteins. However, the rate of increase and the absolute values of the specific radioactivity in LD and HD lipoproteins were identical. Since cholesteryl esters are thought not to exchange between lipoproteins, this observation supports the hypothesis that a result of VLD lipoprotein and chylomicron metabolism is the formation of LD and HD lipoproteins. 5. Results in vivo showed that the free cholesterol of individual plasma lipoproteins does not equilibrate within a period of 24h.     

Effect of serum lipoproteins on the adenylate cyclase activity of rat liver plasma membranes.

Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km.     

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.     

Lipoproteins from the blood and egg yolk of the hen. The transfer of very-low-density lipoprotein to egg yolk and possible changes to apoprotein B

As part of a study of the transfer of proteins and lipids from avian blood to egg yolk, some properties of lipoproteins from the blood of laying hens were compared with those of the low-density lipoprotein (YLP) from egg yolk of the same hens. Although it is known from previous work that particles of the very-low-density lipoprotein (VLDL) of blood are the most likely precursors of YLP, their apoprotein patterns are different, according to electrophoresis and chromatography, with only one protein in common. YLP has the more complicated pattern which does not, however, include apoprotein B (ApoB) the main apoprotein of VLDL. It is suggested that during the transfer of VLDL to yolk, ApoB is cleaved to give smaller yolk apoproteins, especially apovitellenins IV and VI. Some evidence for this suggestion from the similarity of protein digests is presented.     

Isolation and characterization of the major apolipoprotein from chicken high density lipoproteins.

High density lipoproteins were isolated from plasma of white Leghorn hens by ultracentrifugal flotation between densities 1.063 and 1.210 g/ml. After delipidation, the lipid-free proteins were fractionated by chromatography on Sephadex G-150 in urea; one major apolipoprotein was isolated and characterized. From its chemical, physical and immunochemical properties, the major apoprotein from hen high-density lipoproteins has characteristics similar to the major apoprotein of human high density lipoproteins, apoA-I. Thus the hen protein has been designated hen apoA-I. Hen apoA-I has a molecular weight of approximately 28 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Its calculated molecular weight from its 234 constituent amino acids is 26 674. Hen apoA-I differed from its human counterpart by containing isoleucine. Treatment of hen apoA-I with carboxypeptidase A yielded a COOH-terminal sequence of Leu-Val-Ala-Gln. Automatic Edman degradation of the apoprotein gave an NH2-terminal sequence of Asp-Glu-Pro-Gln-Pro-Glu-Leu. Hen apoA-I had a circular dichroic spectrum typical of alpha-helical structures; the calculated helicity was 90%. Goat antisera prepared to hen apoA-I formed precipitin lines of complete identity to the hen apoprotein but lines of only partial identity to human apoA-I. These studies show that the major apoprotein from hen and human high-density lipoproteins have similar properties to each other suggesting a common physiologic function.     

The removal of partially metabolized very-low-density lipoproteins by the perfused rat liver.

1. Donor perfused rat livers were used to prepare VLD (very-low-density) lipoproteins, labelled in their triacylglycerol and protein components with [1-14C]oleic acid and L-[4,5-3H]leucine respectively. Partially metabolized VLD lipoproteins, similarly labelled, were obtained from supradiaphragmatic rats injected with the parent VLD lipoproteins. 2. The triacylglycerol and protein components of the partially metabolized VLD lipoproteins were removed by recipient perfused rat livers at rates much higher than those of the parent VLD lipoproteins. No degradation of the partially metabolized VLD lipoproteins to LD (low-density) lipoproteins occurred during the perfusions. 3. Removal of hepatic lipase from the livers did not significantly affect the rate of removal of the partially metabolized VLD lipoproteins.     

Characterization of the serum lipoproteins and their apoproteins in hypercholesterolaemic guinea pigs.

1. Hypercholesterolaemia was induced in male guinea pigs after 6 days on a chow diet supplemented with 1.6% (w/w) cholesterol and 15% (w/w) corn oil. Both the VLD (very-low-density) and LD (low-density) lipoproteins were increased in cholesterol-fed animals, although the low concentrations of HD (high-density) lipoproteins remained essentially unchanged. LD lipoproteins of d 1.019-1.100 were the major class, accounting for 74% of the total substances of d less than 1.100. 2. Both VLD and LD lipoproteins exhibited alterations in their chemical composition, physical properties and apolipoprotein content. The VLD lipoproteins in cholesterolaemic animals were rich in cholesterol (25.9%), deficient in protein (4.9%) and exhibited electrophoretic mobility greater than that of beta-globulin; their average particle size (64.5 nm) was larger than that in controls (46.3 nm). The LD lipoproteins in animals fed on the experimental diet were also richer in cholesterol (53.1%) and of larger diameter (24.3 nm) than in the control group (41.1% and 21.4 nm respectively). 3. The apolipoprotein-B content of both VLD and LD lipoproteins was elevated in cholesterolaemic animals, particularly in the VLD class, where it represented 74.8% of the total protein moiety. 4. Apo-VLD lipoprotein exhibited an increase from 6 to 19% in its complement of tetramethylurea-soluble apolipoproteins with low electrophoretic mobility (relative mobility less than 0.29); this was primarily accounted for by apolipoproteins characterized by high arginine (7.2 and 6.4% respectively) and glutamic acid (20.1 and 20.0% respectively) contents. 5. By contrast, there was little change in the soluble apolipoproteins of LD lipoproteins in hypercholesterolaemic animals.6. These studies show the response of the guinea pig to dietary fat and cholesterol to be distinct from that elicited by similar stimuli in the rabbit, rat, pig and dog.     

Interaction of rat plasma very low density lipoprotein with lipoprotein lipase-rich (postheparin) plasma.

Incubation of 125I-labeled very low density lipoprotein (VLDL) with lipoprotein lipase-rich (postheparin) plasma obtained from intact or supradiaphragmatic rats resulted in the transfer of more than 80% of apoprotein C from VLDL to high density lipoprotein (HDL), whereas apoprotein B was associated with lipoprotein of density less than 1.019 g/ml (intermediate lipoprotein). The transfer of 125I-labeled apoprotein C from VLDL to HDL increased with time and decreased in proportion to the amount of VLDL in the incubation system. A relationship was established between the content of triglycerides and apoprotein C in VLDL, whereas the amount of apoprotein C in VLDL was independent of that of other apoproteins, especially apoprotein B. The injection of heparin to rats preinjected with 125I-labeled VLDL caused apoprotein interconversions similar to those observed in vitro. The intermediate lipoprotein was relatively rich in apoprotein B, apoprotein VS-2, cholesterol, and phospholipids and poor in triglycerides and apoprotein C. The mean diameter of intermediate lipoprotein was 269 A (compared with 427 A, the mean Sf rate was 30.5 (compared with 115), and the mean weight was 7.0 X 10(6) daltons (compared with 23.1 X 10(6)). From these data it was possible to calculate the mass of lipids and apoproteins in single lipoprotein particles. The content of apoprotein B in both particles was virtually identical, 0.7 X 10(6) daltons. The relative amount of all other constituents in intermediate lipoprotein was lower than in VLDL: triglycerides, 22%; free cholesterol, 37%; esterified cholesterol, 68%; phospholipids, 41%; apoprotein C, 7%, and VS-2 apoprotein, 60%. The data indicate that (a) one and only one intermediate lipoprotein is formed from each VLDL particle, and (b) during the formation of the intermediate lipoprotein all lipid and apoprotein components other than apoprotein B leave the density range of VLDL to a varying degree. Whether these same changes occur during the clearance of VLDL in vivo is yet to be established.     

Characterization of serum lipoproteins of the shark Centrophorus squamous.

1. Blood serum from the shark Centrophorus squamosus (Bonnaterre) was shown to contain VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins. 2. In shape, size and general physical properties, these lipoproteins were very similar to those described for other animals. The VLD lipoproteins were the major components of the mixture, and HD lipoproteins were present at the lowest amount. 3. In addition to the usual lipid components, the shark lipoproteins also contain substantial amounts of hydrocarbon, probably mainly squalene, and monoalkyldiacylglycerols. Only trace amounts of wax ester were detected. 4. The protein moiety of the VLD and LD lipoproteins contained a component which, in its solubility and electrophoretic properties, molecular weight and amino acid composition, resembled the B apolipoprotein of man and other mammals. This accounted for a large part of the total shark apolipoprotein. 5. There were also present smaller amounts of proteins which were soluble in 8M-urea. In their electrophoretic mobility on basic polyacrylamide gel, some of these were like the A and C apoproteins of man. 6. The electrophoretic distribution of the soluble proteins from the VLD and LD lipoproteins resembled that in higher mammals, but in the HD lipoproteins the similarity was less.     

Exclusion of plasma lipoproteins of intestinal origin from avian egg yolk because of their size

Increasing the fat content of the diet increases the proportion of large triglyceride-rich (TGR) lipoproteins (portomicrons) in laying-hen plasma, but has no effect on the size distribution of yolk TGR-lipoproteins. Electromicrographs of the ovarian follicle walls of hens fed a high-fat diet show the presence of numerous portomicron-like particles in the lumen of the thecal capillaries, in the pericapillary spaces and in the theca interna, but portomicrons were absent from the basal lamina, between the granulosa cells and in newly deposited yolk. Most of the lipoprotein lipase activity in the ovarian follicles is associated with the granulosa cells, but total activity in the follicle is very small compared to heart or adipose tissue. The results indicate that the ovarian follicle of the laying-hen specifically excludes lipoproteins of intestinal origin from yolk, most probably because they are too large to pass through the connective tissue matrix of the basal lamina. The low lipoprotein lipase activity of the ovarian follicle, together with its distribution within the follicle wall, indicates that the ovarian follicles make little contribution to catabolism of circulating portomicrons.     

Does vitellogenin inhibit lipoprotein lipase in the laying hen?

Vitellogenin isolated from laying-hen plasma strongly inhibited chicken adipose-tissue lipoprotein lipase in vitro, but inhibition was reduced or prevented by Ca2+ and Mg2+ ions and by partial dephosphorylation. Plasma from blood collected from laying hens using EDTA as anticoagulant was a potent inhibitor of lipoprotein lipase, but serum from laying hen blood caused inhibition only when dilute or after addition of EDTA. Heparin reduced or abolished the inhibition of lipoprotein lipase by plasma, serum and purified vitellogenin. The results suggest that inhibition of lipoprotein lipase by vitellogenin requires the presence of charged phosphate groups on vitellogenin and an unoccupied heparin-binding site on the enzyme. Neither condition is likely to occur in the laying hen in vivo.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.