Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein.

Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.     

Studies on pig serum lipoproteins. VI. Surface charge of very low density lipoprotein.

The surface electric charge of pig serum very low density lipoprotein (VLDL) is described. By isoelectric focusing VLDL was separated into at least 3 fractions having different isoelectric points and polypeptide distributions. The ultracentrifugal and electron microscopic results indicate that the VLDL was not drastically denatured by Ampholine.     

Studies on pig serum lipoproteins. I. Separation and properties of low-density lipoproteins.

The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1.     

Flotation equilibrium of serum low density lipoproteins

The molecular weight of human serum low density lipoprotein (LDL) was determined for the first time by sedimentation equilibrium or, more accurately, flotation equilibrium, in high concentration of KBrNaBr containing Tris-Cl buffer plus EDTA (density = 1.20 – 1.49 g/ml). Assuming both the molecular weight and the partial specific volume were unknown, the results at different densities gave a value of 2.87 ± 0.12 × 10⁶ for the molecular weight and 0.965 ± 0.014 ml/g for the partial specific volume.     

Studies of the cyanogen bromide fragments of the apoprotein of human serum low density lipoproteins.

The apoprotein of human serum low density lipoproteins was reduced and carboxymethylated and then cleaved by cyanogen bromide (CNBr). The peptides which were produced from this cleavage (90% yield, based upon loss of methionine) were resolved by SDS polyacrylamide gel electrophoresis into 10 major bands, each having an amino acid composition very similar to that of intact reduced and carboxymethylated LDL apoprotein. The fractionation of the CNBr fragments by preparative gel filtration was dependent upon the nature of the eluting solvent. NH4OH and SDS solvents eluted all of the material in the void volume. In 6 M guanidinium chloride solvents several peaks were, however, resolved, each having an amino acid composition similar to that of the unfractionated products. Whereas no NH2-terminal was detected in reduced and carboxylmethylated LDL apoprotein, automated Edman degradation of the protein following treatment with CNBr revealed the presence of several NH2-termini. The results suggest that LDL apoprotein may be made of segments of, at least, very similar amino acid composition and that both the protein itself and derivative fragments have a great tendency to aggregate even in denaturing solvents.     

Studies on the binding of 1-anilino-8-naphthalene sulfonate to very low density and high density himan serum lipoproteins.

Very low density and high density lipoproteins have been isolated from human plasma and their interaction with 1-anilin0-8-naphthalene sulfonate has been studied under different conditions of pH and added salt. Intrinsic fluorescence of bound 1-anilino-8-naphthalene sulfonate was higher for high density lipoproteins then for very low density lipoproteins, but was unaffected by salt in both systems. Binding of 1-anilino-8-naphthalene sulfonate by both these lipoproteins was saturable and was higher in the presence of added NaCl or CaCl2, Ca2+ having a greater effect than Na+ in enhancing fluorescence. The binding data were analyzed by Scatchard plots; the number of binding sites and the affinity of 1-anilino-8-naphthalene sulfonate for the site increased with increasing salt concentration. Fluorescence pH curves were similar to those published for phospholipids. From these and previous observations it is suggested that the phospholipids probably represent the major binding sites for 1-anilino-8-naphthalene sulfonate.     

Secondary structure in very low density and intermediate density lipoproteins of human serum.

We have studies the secondary structures of the protein moieties of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) of human serum by circular dichroism (CD). Two potential complications in the application of this technique to lipoproteins have been evaluated. First, using chronographic potentiometry in CD measurements of VLDL fractions of different mean particle diameters, we have analyzed statistically the CD signals in order to define the limits imposed by light scattering with respect to both particle diameter and wavelength. We found that CD measurements can be made to as low as 210 nm on particles of 520 A or smaller, and to 194 nm on particles of 450 A and below. Second, we have evaluated the CD contribution of lipid chromophores. Despite the high ratio of lipid to protein, the relative CD effect of the lipids is smaller than for low density lipoproteins (LDL). due to the extremely small ellipticity of natural VLDL triglycerides. Thus, CD measurements can be obtained with confidence on the preponderant bulk of normal VLDL. For the first time we report the CD spectra of human VLDL and IDL. In contrast with human LDL and the lipoproteins of the hypercholesterolemic rabbit, the entire CD SPECTRUM OF HUMAN VLDL shows increased ellipticity with decreasing temperature, which is completely reversible. We have found that the protein moieties of human VLDL and IDL contain substantially more helix (approximately 50%) than does that of human LDL.     

Guinea pig low density lipoproteins: structural and metabolic heterogeneity

The structural and metabolic heterogeneity of low density lipoproteins (LDL, d 1.024-1.100 g/ml) has been investigated in the guinea pig. Two LDL subfractions, of d 1.024-1.050 and 1.050-1.100 g/ml, respectively, were isolated by sequential ultracentrifugation; while both were enriched in cholesteryl ester and apoB-100, the former was heterogeneous displaying three particle size species of diameters 26.9, 25.6, and 24.7 nm, whereas the denser subfraction was relatively homogeneous containing a single, smaller species (diam. 23.6 nm). The fractional catabolic rates (FCR) of the two LDL subfractions were alike (approximately 0.090 pools/hr) in the guinea pig in vivo. After modification of each subfraction by reductive methylation, the FCRs were reduced similarly and indicated that 70-80% of degradation occurred via the cellular LDL receptor pathway. However, the intravascular metabolism of these LDL subfractions, determined from the radioactive content of density gradient fractions as a function of time after injection of radiolabeled native or chemically modified LDL, tended to be distinct. Thus, while radiolabeled apoB-100 in the lighter subfraction maintained the initial density profile up to 48 hr, the radioactive profile of its methylated counterpart changed, the proportion of radioactivity in the lighter gradient fractions (d 1.027-1.032 g/ml) increasing while that in the denser (d 1.037-1.042 g/ml) fractions diminished. A more marked transformation occurred in LDL of d 1.050-1.100 g/ml, in which the radioactive profile shifted towards lighter particles of the d 1.024-1.050 g/ml species; this shift was partially dependent on the LDL receptor, since it was more pronounced in the methylated subfraction. Furthermore, a net increase in the radioactive content of gradient subfractions 7 to 9 (d 1.032-1.042 g/ml) was found 10 hr after injection of methylated LDL of d 1.050-1.100 g/ml, at which time the bulk of LDL radioactivity had been removed from plasma. Several mechanisms, acting alone or in combination, may account for these findings; among them, some degree of transformation of dense to lighter LDL species appears a prerequisite. In conclusion, our data attest to the structural heterogeneity of circulating LDL in the guinea pig, and suggest that the intravascular processing and metabolism of LDL particle subspecies is directly related to their structure and physicochemical properties.     

Studies on oxidized low density lipoproteins. Controlled oxidation and a prostaglandin artifact

Low density lipoproteins (LDL), isolated by ultracentrifugal flotation, were oxidized (LDLOXID) slowly during dialysis against 0.15 M NaCl and subsequent incubation in 96% air-4% CO2 at 37 degrees C. Butylated hydroxytoluene prevented LDL oxidation. LDL preparations from different sera were oxidized at different rates and the degree of lipid peroxidation was controlled by varying the incubation time. Mild oxidation did not alter the electrophoretic mobility of the LDLOXID preparations. LDLOXID contained lipid peroxides in neutral lipids, had increased amounts of lysophosphatidylcholine, and contained a number of complex oxidation products that were generated from the oxidation of free fatty acids. These oxidation products included large amounts of soluble material that cross-reacted with antibodies to PGE2 but not 6-keto-PGF1 alpha. The amount of cross-reacting material was proportional to the degree of lipid peroxidation. Cross-reacting material in LDLOXID preparations was evidently formed from the oxidation of free fatty acids released from LDL, since cross-reacting material was also formed when a synthetic fat emulsion was oxidized in the presence of free arachidonic acid.     

Metabolism of very low density lipoproteins in the pig. An in vivo study.

1. The metabolism of apolipoprotein B (apoB) was investigated in pigs injected with [125I]very low density lipoproteins (VLDL) to determine to which extent the two distinct low density lipoprotein subclasses (LDL1 and LDL2) derive from VLDL. 2. The lipoproteins were isolated by density gradient ultracentrifugation and the transfer of radioactivity from VLDL into LDL1 and LDL2 apoB was measured. 3. Only a minor portion of VLDL apoB was converted to LDL1 (7.7 +/- 3.2%) and LDL2 (3.6 +/- 1.5%), respectively. Thus, we conclude that the major portion of LDL, especially LDL2, is synthesized independently from VLDL catabolism.     

Synthesis and release of low density lipoproteins by the isolated perfused pig liver.

In previous studies, we have shown that a relatively large amount of low density lipoproteins is released into the perfusate during isolated pig liver perfusion. The present studies were done to determine the source of these lipoproteins. Breakdown of the very low density lipoproteins to low density lipoproteins by the perfusion apparatus or by hepatic catabolism was excluded by adding 125I very low density lipoproteins to the perfusate in the presence and absence of a liver and then measuring the radioactivity in the low density lipoprotein fraction after rate-zonal ultracentrifugation. Release of preformed low density, lipoproteins from the liver was investigated by injecting iodine-labeled low density lipoproteins in vivo several hours prior to perfusion of the liver and then measuring the release of labeled low density lipoproteins into the perfusate. It was shown that intact labeled low density lipoproteins were released by the perfused liver. De novo synthesis of the low density lipoproteins was established by measuring the incorporation of [1-14C]leucine into this lipoprotein fraction. The radioactivity in the low density lipoprotein fraction increased with time and accounted for 20 to 25% of the total radioactivity incorporated into all the lipoprotein fractions. The incorporation of [1-14C]leucine into the low density lipoproteins was confirmed by rate-zonal analysis. We conclude that the low density lipoproteins in the perfusate from pig liver perfusions were derived mainly from a preformed liver pool, but also partly from de novo synthesis by the liver.     

Studies on the protein composition of human serum very low density lipoproteins: demonstration of the beta 2-glycoprotein-I.

Human serum VLDL isolated by polyanion precipitation and ultracentrifugation have been delipidated with ethanal/diethyl ether. By electrophoresis in 10% polyacrylamide gels containing 8M urea, we found a protein which comigrated with apolipoprotein E. This protein was purified by column chromatography and turned out to be identical with beta 2-glycoprotein-I, the serum factor which is necessary for the precipitation of triglyceride-rich lipoproteins with sodium decyl sulfate or sodium dodecyl sulfate. Upon analytical isoelectric focusing, beta 2-glycoprotein-I gave four major bands in the pH region 5.7--6.6. All four bands gave an immunochemical reaction of identity with a monospecific antiserum. From its unique amino acid composition we conclude that beta 2-glycoprotein-I is distinct from all apolipoproteins described previously in the literature.     

Studies on the composition of pig serum lipoproteins. Isolation and characterization of different apoproteins.

1. Different lipoprotein density fractions from pig serum were isolated by phosphotungstate precipitation followed by purification in the preparative ultra-centrifuge. 2. The protein part of very low density lipoproteins was composed of approximately 52 percent lipoprotein B apoprotein and the rest of lipoprotein C II apoprotein and other as yet unidentified peptides. 3. The protein moiety of low density lipoproteins consisted primarily of lipoprotein B apoprotein (over 95 percent); the amino acid compositions of lipoprotein B apoprotein of very low and low density lipoproteins were practically identical. 4. The predominant polypeptide of pig serum high density lipoproteins exhibited an amino acid composition and a molecular weight very similar to human liprotein A I apoprotein. In contrast to human lipoprotein A I apoprotein, the apoprotein from pigs was found to release leucine first followed by alanine, threonine, and lysine upon incubation with carboxypeptidase A. 5. In pig serum the major lipoprotein C apoprotein was found to be a polypeptide similar in amino acid composition to lipoprotein C II apoprotein from human serum. The molecular weight of this polypeptide is approximately 8000. Incubation experiments with carboxypeptidase A indicate serine to be the most likely C-terminal amino acid.