Bovine Serum High Density Lipoprotein Research Articles

Bovine apolipoproteins C: I. Isolation and spectroscopic investigations of the phospholipid binding properties

Seven low-molecular weight proteins of the C class of apolipoproteins have been isolated from bovine serum high density lipoprotein. Amino acid analysis has shown five of these to be equivalent to the apolipoproteins previously described (Lim C.T. and Scanu A.M. (1976) Artery 2, 483–496). A spectroscopic examination of these proteins in the presence of increasing amounts of l-α-dimyristoyl phosphatidylcholine single-bilayer vesicles indicates that all bovine apolipoproteins C exhibit changes in secondary and tertiary structure as shown by intrinsic fluorescence intensity, wavelength, and polarization changes, and increases in α-helical content as seen by circular dichroism. Evidence is presented to show that bovine apolipoproteins C, like all human apolipoproteins of the A and C classes, can cause phospholipid multilamellar liposomes to disrupt and/or rearrange into a smaller complex which scatters less light. This paper details the screening of the bovine apolipoproteins for their phospholipid binding properties, whereas the following paper will examine the nature of their complexes with phospholipid in more detail. Together, these papers represent the first investigation of protein-lipid interactions involving any nonhuman apolipoproteins C.

Interaction of bovine serum high density lipoprotein with mixed vesicles of phosphatidylcholine and cholesterol.

The interaction of sonicated, small vesicles of egg phosphatidylcholine and cholesterol (2:1, mol/mol) with bovine high density serum lipoproteins was examined in terms of lipid transfer between both types of particles and the resulting changes in lipoprotein structure. Saturation of high density lipoprotein preparations with vesicle lipids gave final lipoprotein particles with essentially unchanged protein content and composition, unchanged cholesterylester and nonpolar lipid content, but with markedly increased phospholipid content (59% increas by weight) and moderately increased cholesterol content (20% increase by weight). The lipoproteins enriched in lipid were relatively uniform, spherical particles, 110 +/- 3.6 A in diameter (6 A larger than the original lipoproteins); they had a markedly decreased intrinsic protein fluorescence, a red-shifted fluorescence wavelength maximum, and more fluid lipid domains. These results indicate that the direct addition of excess lipids from membranes or other lipoproteins is a possible mechanism for lipid transfer to high density lipoproteins. Also they suggest a structural flexibility of high density lipoproteins that allows the addition of significant amounts of surface components.

Kinetics and mechanism of phosphatidylcholine and cholesterol exchange between single bilayer vesicles and bovine serum high-density lipoprotein

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetics and mechanism of phosphatidylcholine and cholesterol exchange between single bilayer vesicles and bovine serum high-density lipoproteinAna Jonas and Gregory T. MaineCite this: Biochemistry 1979, 18, 9, 1722–1728Publication Date (Print):May 1, 1979Publication History Published online1 May 2002Published inissue 1 May 1979https://doi.org/10.1021/bi00576a014RIGHTS & PERMISSIONSArticle Views78Altmetric-Citations69LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (829 KB) Get e-Alerts Get e-Alerts

Effects of triton X-100 treatments on the composition and activities of membrane vesicles from pigeon erythrocytes

Membrane vesicles were prepared from pigeon erythrocytes. The effect of various treatments on the ability of these vesicles to trap and transport glycine was measured. Most of the work concerned the effects of the nonionic detergent, Triton X-100 (Triton). Triton inhibits the capacity of membrane vesicles to trap and transport glycine. The effects of Triton depend on temperaature and pH. At neutral pH and 41°C, the half-inactivating dose of Triton is 6 μl/g wet weight of membrane, while at neutral pH and O°C it is approx. 60 μl/g. At pH 8.5–8.8 and 0°C the half-inactivating dose is 15–20 μl/g. The Triton effect depends on the ratio of Triton to membrane, not the Triton ‘concentration’ in the aqueous phase. Protein is released from the membrane by Triton treatment at 0°C. At pH 8.5–8.8, the dose-response curve for protein release is very similar to the dose-response curve for inhibition of the capacities to trap and take up glycine. All three curves have steep and shallow regions, respectively below and above a Triton dose of 15 μl/g. The protein released by Triton treatment at 0°C is largely the lower molecular weight classes and the maximum protein release is 50–60% of the total membrane protein. The percent of this protein class released by a given Triton dose equals the percent inhibition of the capacity to trap glycine. Triton is bound by the membranes. In the dose range of 0–10 μl Triton/g wet membrane, about half of the added Triton is bound. This bound Triton is not readily removed by washing. Like protein release and inhibition of trapping and transport capacities, the binding process has two phases, one above and one below a dose of 15 μl/g. The membrane is saturated with Triton at a dose of 15 μl/g with 4.5 μl Triton bound/g wet weight, corresponding to 85–90 μl bound/g remaining membrane protein. Bovine serum high density lipoprotein efficiently removes bound Triton from the membrane. By removing Triton bound at 0°C with lipoprotein, the effects of Triton at 0 and 41°C could be distinguished and the time course of Triton action at 0°C could be measured. Triton action was nearly complete by 10 min at 0°C. Possible modes of action of Triton on these membranes are discussed. At least part of the action of Triton can be described as a process in which membrane proteins are partitioned between the membrane phase and an extramembranal Triton micelle phase.

Interaction of phosphatidylcholine with bovine serum albumin. Specificity and properties of the complexes

Phosphatidylcholine dispersed on Celite was rapidly solubilized by neutral bovine serum albumin solutions. Stable protein-lipid complexes were isolated by Agrose gel filtration or by ultracentrifugal flotation in high density solvents, and the physicochemical properties of the complexes were investigated in terms of the stoichiometry of binding, effect of fatty acid ligands on phosphatidylcholine binding, effect of high ionic strength on the stability of the complexes, intrinsic fluorescence and circular dichroism spectra, and sedimentation velocity coefficients. Complexes containing from 2 to 30 phosphatidylcholine molecules per protein molecule were observed; however, no saturation of binding sites could be detected in this range of molar ratios. Oleic acid binding by serum albumin prevents interaction of the protein with phosphatidylcholine, indicating possible competition of these ligands at low contents of the phospholipid. For molar ratios of up to 10 phosphatidylcholine molecules per serum albumin, binding is primarily due to hydrophobic interactions that have no effect on the overall shape and secondary structure of the native protein except for local modifications at tryptophan residues, whose fluorescence becomes quenched and blue shifted on phosphatidylcholine binding. Similar phosphatidylcholine uptake experiments performed with a series of globular proteins indicated that the lipid extraction from Celite surfaces is a non-specific process, accelerated by several other proteins (e.g. aldolase, egg albumin, chymotrypsinogen, soybean trypsin inhibitor, and the major apolipoprotein from bovine serum high density lipoprotein). Formation of stable protein-lipid complexes, however, was only observed with bovine serum albumin, which in contrast to the other proteins is known to have high affinity binding sites for anions with hydrophobic side chains.

Fluidity of the lipid phase of bovine serum high density lipoprotein from fluorescence polarization measurements

The microviscosity of the lipid phase of bovine serum high density lipoprotein was determined by fluorescence polarization measurements on a lipophilic probe (1,6-diphenyl-1,3,5-hexatriene) dissolved in the lipoprotein. At 25°C the average microviscosity was 6.1 ± 0.5 poise, and the activation energy calculated from a plot of log η versus 1 T was 13±3Kcal/mole. A constant slope for the Arrhenius plot from 0 to 46°C indicated no apparent phase transitions in this temperature range. Comparison of the present results with reported microviscosity values for rat lymphocyte membranes and liposomes [Shinitzky and Inbar (1974) J. Mol. Biol. 85 , 603] indicates a more rigid environment of the probe in the high density lipoprotein system fluidity of the lipid appears to be considerably decreased in the lipoprotein relative to organic solvent or oil solutions of lipids, probably as a result of the anisotropic environment of the probe, high total cholesterol, and presence of protein in these particles.

Isolation and partial characterization of the major apolipoprotein component of bovine serum high density lipoprotein

The delipidated protein component of bovine serum high density lipoprotein was fractionated by gel filtration on a Sephadex G-150 column (equilibrated with buffer containing 6 M urea) into three fractions: I, II and III. Fractions I and II together constitute 88% of all the protein weight of bovine high density lipoprotein, whereas fraction III accounts for the remaining 12%. Analysis of the fractions by sodium dodecyl sulfate-polyacrylamide electrophoresis reveals that fraction I consists mostly of aggregated forms of fraction II and some higher molecular weight species, probably irreversible aggregates of fraction II. The irreversible aggregates are apparently formed during the delipidation procedure or upon aging of the lipoprotein. The major protein component of the high density bovine lipoprotein is found in fraction II; it has a molecular weight of 27 000 ± 1500 and appears to be homogeneous by several physicochemical criteria. The amino acid composition of fractions I and II are essentially identical; their spectral properties, including absorption, fluorescence, and circular dichroism spectra, are similar; however, fraction I appears to contain traces of oxidized lipid and more secondary, α-helical organization than fraction II. By comparison with the intact lipoprotein, which contains about 65% of α-helical structure, fractions I and II have diminished α-helical organization, 55% and 43%, respectively. Fraction III, on sodium dodecyl sulfate-polyacrylamide electrophoresis, separates into two protein bands of equivalent intensity, having molecular weights around 13 000 and 11 000. Fraction III is markedly distinct from the other two, in amino acid composition and spectral properties, especially in its red-shifted fluorescence and very low content of α-helical structure. The protein composition of bovine serum high density lipoprotein is compared with recently published results for high density lipoprotein apoproteins of man, chimpanzee, rhesus monkey, pig and rat. Similarities and differences are discussed in terms of possible evolutionary and functional factors.

Self-association of the major protein component of bovine serum high density lipoprotein

The major protein component of bovine high density lipoprotein was investigated in solution by fluorescence polarization and ultracentrifugal techniques. A fluorescent derivative of this protein with 1-dimethylaminonaphthalene-5-sulfonyl chloride was employed in the fluorescence experiments. Over the concentration range from 5·10 −7 M to 5·10 −4 M of the protein monomer at pH values from 2 to 11 and ionic strengths from 0.03 to 2.0, at 23 °C, the major protein of bovine high density lipoprotein apoprotein (Apo-HOL-I) was found to exist in a stable aggregated form. The aggregate was not affected by dioxane additions of up to 20% nor by Triton X-100 to 0.2%, but dissociated readily in the presence of 0.07% sodium dodecylsulfate or 6 M urea. At concentrations below 5·10 −7 M, dissociation of the protein aggregate started spontaneously and continued down to 10 −8 M, the lowest measurable concentration. Several physicochemical properties of the major protein of bovine high density lipoprotein were determined in the stable aggregate form. Molecular weight was 104 000 from ultracentrifugal analysis and 80 000 from gel-filtration. Rotational relaxation time was 115 ns at 25 °C, and s 0 20,w was 4.78 s. The results suggest very strong protein-protein interactions ( K d < 10 −7 M) that are not electrostatic in nature. Hydrophobic interactions of a magnitude that could be affected by 20% dioxane or 0.2% Triton X-100 detergent are also excluded. There is saturation of the interaction sites by the aggregation of a few protein monomer units possibly to form a tetramer which is moderately asymmetric (1:4 axial ratio, assuming an ellipsoid of revolution) and relatively rigid. The strong protein-protein interactions in this pure apolipoprotein suggest the possibility of competition of inter-protein associations with protein-lipid interactions in in vitro lipid binding or lipoprotein reconstitution experiments.

Studies on the Structure of Bovine Serum High Density Lipoprotein Using Covalently Bound Fluorescent Probes

Abstract Fluorescence polarization, fluorescence lifetime, and sedimentation velocity measurements on fluorescent conjugates of bovine serum high density lipoprotein with 1-dimethylaminonaphthalene-5-sulfonate and 1-pyrenebutyrate provide considerable information on the structure of the lipoprotein, including its rotational diffusion, size, shape, flexibility, and response to substances which alter the structure of biological membranes. Bovine serum high density lipoprotein has an average rotational relaxation time of 570 ns at 25°; it is a relatively rigid, spherical particle with some local flexibility at the level of protein side chains and the individual lipid components. In the presence of increasing amounts of 1-octanol, the lipoprotein first expands and finally becomes disrupted. The structural changes are qualitatively similar to those produced by alcohol anesthetics on biological membranes.