Anton Holasek

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.

Studies on the structure of lipoprotein A of human high density lipoprotein HDL3: the spherically averaged electron density distribution.

1. Introduction In a previous article [l] we gave a first report on the experimental results obtained by X-ray small angle scattering of the LpA fraction of human plasma high density lipoprotein HDL3 in 0.15 M NaCl solution. The shape of the scattering curve showing three characteristic side maxima reflected a high structural regularity of the molecule. In the present paper we give a more detailed analysis of the scattering curve mentioned above, as well as additional results from X-ray small angle scattering experiments in 36% sucrose solution. The results are strongly in favour of a spherical molecular model of 96 A diameter consisting of an inner part of low elec- tron density surrounded by a high electron density shell.

The intestinal absorption of glycerol trioctadecenyl ether.

Abstract 1. 1. Glycerol tri- cis -9-octadecenyl ether is used as a model substance for triolein in studying the intestinal absorption of non-hydrolysed triglycerides. 2. 2. [ 14 C]Glycerol trioctadecenyl ether was fed to rats provided with a thoracic duct fistula and to non-operated rats. The radioactivity of the lipids of lymph, some organs and the whole carcass was determined. 3. 3. The very poor absorption of [ 14 C]glycerol trioctadecenyl ether confirms the suggestion of others that the absorption of intact triglycerides must be very poor.

X-ray small angle scattering of human plasma high density lipoprotein LpA from HDL2: application of a new evaluation method.

In their recent study, Shipley et al. [ 1 ] presented data on the structure of HDL, as obtained from X-ray small angle scattering. The results were in good qualitative agreement with the results of our previous studies on LpA from HDL3 [2,3] indicating a common molecular architecture for these two species of human plasma high density lipoproteins. However, the proposed model for HDL, was based only on comparison of theoretical and experimental scattering curves, obtained from experiments in solvents of one single electron density. In this case where a non homogeneous electron density within the molecule must be assumed a priori, the fit of the data is only to be considered as a necessary but not sufficient condition. Therefore we extended our studies on lipoproteins also to the HDL, species. This article is a report on the results which were obtained from X-ray small angle scattering of LpA from HDL, in different solvents. For the translation of the scattering data into real space information we employed for the first time a novel procedure

X-ray small angle scattering on human plasma lipoproteins☆

Abstract Two immunochemically defined lipoprotein fractions from human plasma (LpB from Low Density Lipoprotein and LpC from High Density Lipoprotein HDL 2 ) were studied by X-ray small angle scattering. The molecular weights were found to be 2.4 × 10 6 for LpB, and 4.6 × 10 5 for LpC. In both cases the scattering curves indicated spherical symmetry of the particles. The radial electron density distributions were obtained by Fourier transformation. LpB showed an overall radius of approx 120 A, and three electron density maxima around 30, 65, and 105 A. LpC showed a low electron density core and a high electron density shell extending from approx 55 to 70 A radii.

Small angle X-ray scattering of LpA, the major lipoprotein family of human plasma high density lipoprotein HDL3

Most of the present knowledge of size and shape of human plasma high density lipoproteins (HDL) is based on their visualization by electron microscopy using negative staining techniques [ 1,2]. These experiments indicate a subunit structure for both the classes of high density lipoproteins, HDb and HDL3. Unfortunately the resolution has been limited to about 30 A, which did not allow to give precise dimensions of the subunit structure. Moreover, these pictures revealed a great variety of possible arrangements for the subunits within the particles of each class. The assumption of a subunit structure of the constituent protein moiety was strongly supported by delipidation experiments. Several groups of investigators have shown [3-51, that the apoprotein can be fractionated into nonidentical polypeptides. It is a well established fact, that lipoprotein density classes are heterogeneous with respect to their chemical, immunochemical and physicbchemical properties [6-81. In fact there does not seem to exist any density fraction, from which a single, homogeneous lipoprotein fraction can be isolated solely by ultracentrifugation. We therefore applied additional purification procedures to isolate a lipoprotein A (LpA) preparation from HDLJ fraction of human serum, homogeneous with respect to its immunochemical and physicochemical behaviour. This paper is a first report of our X-ray small angle scattering experiments on LpA. The results given are confined to the most unambiguous molecular parameters as the radius of gyration, the molecular weight and the particle volume. The results indicate a subunit structure for LpA.

In vivo studies on the binding sites for lipoprotein (a) on parenchymal and non-parenchymal rat liver cells

The direct correlation between lipoprotein (a) (Lp(a)) concentrations and atherosclerosis stimulated us to investigate the in vivo interaction of Lp(a) with the liver and the various liver cell types. In untreated rats the serum decay of Lp(a) is comparable to that of LDL. By estrogen treatment the interaction of LDL with parenchymal liver cells is increased 17‐fold whereas only a 2‐fold effect on Lp(a) is found. The decay of Lp(a) in estrogen‐treated rats is slower than for LDL. The data indicate that Lp(a) in vivo shows a less efficient interaction than LDL with the estrogen‐induced apo‐B,E receptor on parenchymal liver cells. It is suggested that the inability of Lp(a) to interact efficiently with the LDL removal system of the liver might be related to its atherogenic action.

Factors affecting the conversion of high-density lipoproteins: experiments with pig and human plasma

The conversion of pig high-density lipoproteins (HDL) (mainly HDL3) to fractions of lower densities was studied by incubating pig plasma for 24 h at 37 degrees C in the presence and absence of lipoprotein lipase from bovine milk, lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles (very-low-density lipoproteins (VLDL) or Intralipid). The results can be summarized as follows. In the presence of lipoprotein lipase and at a VLDL/HDL mass ratio of 2, the F-1.210 of pig HDL was shifted from 3.3 to 4.2, which is characteristic for human HDL2. This shift was caused by the excessive increase in the free fatty acid content in HDL. If 50 g/l of bovine serum albumin were added prior to incubation, the flotation rate of HDL remained in the HDL2a region. If lecithin:cholesterol acyltransferase was active in fasting pig plasma during incubation, we observed only a negligible increase of F-1.210 in HDL. If pig lipoproteins were incubated with human lipoprotein-free serum as a source of cholesteryl ester transfer activity, a slight increase in the flotation rate of HDL was observed, which was amplified in the presence of active lecithin:cholesterol acyltransferase. Pig HDL was converted to a fraction with F-1.210 of 4.2, which is typical for human HDL2, only if active lecithin:cholesterol acyltransferase, cholesteryl ester transfer protein and triacylglycerol-rich particles were present in the incubation mixture. From our results we also concluded that apolipoprotein A-II plays no role in the HDL2 formation.

Molecular parameters of Lp(a)-lipoprotein by light scattering

Lp(a) lipoprotein is thought to be an addi- tional risk factor for coronary heart disease [l-3]. Papers published up to now have been concerned with its electrophoretical mobility [4,5], its chemical com- position [6,7] and its physiological role [l-3]. Con- cerning the size of the particle, only disagreeing information has been reported [8]. Serum concentra- tion of that lipoprotein differs from man to man, and Harvie and Schultz were the first to demonstrate that Lp(a) is expressed by a quantitative genetic trait [9,10]. A diet enriched by cholesterol did not influence the serum Lp(a) level, while LDL increased markedly [11,12]. As pointed out [13,14] Lp(a) reacts very sensitively on addition of Ca2+. The aim of this work was to provide for this lipo- protein the values for molecular weight (a,) diffu- sion coefficient (D), radius of gyration Cs;) and hydrodynamic radius

Isolation of human serum low-density lipoproteins with the aid of an immune-specific adsorber.

Human serum lipoproteins containing B-protein have been isolated using an immunoadsorber. Bromoacetyl cellulose was combined with pure antibodies to low density lipoprotein (LDL) and an immunoadsorber of high capacity was obtained. With 1 g of this immunoadsorber all LDL and very low density lipoprotein (VLDL) from 30 ml pooled human serum were adsorbed and then eluted with glycine-HCl buffer pH 3.2 at 0 C. The isolated lipoproteins were investigated by electrophoresis, immunodiffusion and ultracentrifugation, and found to be identical to LDL+VLDL isolated by ultracentrifugation.