Electrophoretic separation of high-molecular hydrophobic proteins: apolipoprotein B and other protein constituents of low-density lipoproteins

Apolipoprotein B (apoB) and 40 different protein standards were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under various conditions. The system selected is capable of fractionating well the apoB and its high-molecular-weight species formed after treatment of serum lipoproteins by malonic dialdehyde. The basis for good resolution of protein bands is the use of complex gels combining in one block the areas with constant (T = 3 and 10%) or continuously changing (gradient gel with T = 3-6%) concentrations of acrylamide. The procedure allows one to create the optimal conditions for simultaneous separation of both high- and low-molecular-weight polypeptides. The method was used to determine the relative molecular mass (Mr) of apoB. When apoB was isolated in the presence of proteolytic inhibitors, its Mr was found to be equal to 540,000 +/- 9500 or 500,000 +/- 5700, depending on the linear regression equation applied. The results are in good accordance with values calculated from the amino acid sequence of mature apoB.     

Electrophoretic separation of plasma lipoproteins in agarose gel

A method has been developed for the separation of serum or plasma lipoproteins by electrophoresis in an agarose-agar gel mixture. The gel is applied to the surface of a thin polyester photographic film strip. With minor alterations in technique either single samples on individual strips or many samples on one large sheet may be processed. After fixation and dehydration the transparent film is stained with Sudan Black B and washed with water. The finished electrophoretogram can be obtained in 5 hr and consists of widely separated bands of lipoprotein fractions on a colorless transparent background, ideally suited for scanning with a densitometer. Plasma samples from different subjects show pre-beta lipoproteins of different mobilities. An effect of gel concentration on the extent of lipoprotein migration is demonstrated. The clearcut separation of lipoproteins by this method will facilitate the classification of hyperlipoproteinemias and improve quantitative estimates of lipoprotein distribution.     

Evidence that apoB-100 of low-density lipoproteins is a novelsrc-related protein kinase

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of thesrc protein kinase family.     

Antioxidant effect of high-density lipoproteins in the peroxidation of low-density lipoproteins

It has been shown that in the solution of low density lipoproteins (LDL) during their incubation at 37 degrees C the turbidity and concentration of malondialdehyde was increased, as compared to that observed at 4 degrees C. Both parameters were slowed down by the addition of high density lipoproteins (HDL) into the medium. The protective effect of HDL depended on the time of incubation and the concentration of HDL added. Delipidated HDL had no effect. Similar action of HDL was established in the experiments where the peroxidation in LDL was induced by the xanthine-xanthine oxidase. The data obtained demonstrate that HDL possess an antioxidant property that may play an important role in their antiatherogenic action.     

Sequestration of aggregated low-density lipoproteins by macrophages

PURPOSE OF REVIEW: Evidence suggests that much of the LDL in atherosclerotic plaques is aggregated. Aggregation of LDL could be an important factor that determines how this lipoprotein is metabolized by plaque macrophages and the fate of aggregated LDL cholesterol within plaques. This review discusses a novel endocytic pathway by which macrophages process aggregated LDL. RECENT FINDINGS: Recently, it has been shown that aggregated LDL can be sequestered in macrophage surface-connected compartments and plasma membrane invaginations by a process termed patocytosis. In contrast to rapid degradation of LDL and aggregated LDL taken up by macrophages through pinocytosis and phagocytosis, respectively, aggregated LDL sequestered in macrophages undergoes only limited degradation. Macrophages can disaggregate and release sequestered aggregated LDL by activating plasminogen to plasmin. Plasmin degrades LDL apolipoprotein B sufficiently to disaggregate the aggregated LDL, releasing it from the macrophage surface-connected compartments. In contrast, activating macrophages with phorbol-myristate-acetate stimulates degradation of aggregated LDL and inhibits plasminogen-mediated release of the aggregated lipoprotein from macrophage surface-connected compartments. SUMMARY: Macrophage sequestration of aggregated LDL is a unique endocytic pathway relevant not only to the processing of aggregated LDL in atherosclerotic plaques but also for the processing of other materials, such as hydrophobic particles that trigger this endocytic pathway. Macrophage sequestration of aggregated LDL can result in different fates for the aggregated LDL, depending on the state of macrophage activation and the functioning of the plasminogen-based fibrinolytic system. Patocytosis of aggregated LDL should be considered in addition to phagocytosis as a possible uptake pathway in studies of macrophage processing of aggregated LDL.     

Evidence that apoB-100 of low-density lipoproteins is a novelsrc-related protein kinase

Protein-tyrosine kinases of signal transduction pathways occur and function intracellularly. In contrast, the low-density lipoprotein (LDL) particle circulates in plasma, where its function is to solubilize and transport lipid. Recently, several reports showed that LDL may have a role in signal transduction. We have identified a region in the apoB-100 primary structure which shows similarity to Src-homology-1 (SH1) domains, the kinase region of protein-tyrosine kinases. Results obtained in protein kinase assays of highly purified LDL showed that only the apoB-100 was phosphorylated, suggesting that apoB-100 has the capacity to undergo autophosphorylation like known protein-tyrosine kinases. Phosphorylation was not observed for any other apolipoprotein in LDL or for any component of high-density lipoprotein and lipoprotein [a]. Our results suggest that apoB-100 may be a novel and functional member of thesrc protein kinase family.     

Preparative isoelectric focusing of human serum very low-density and low-density lipoproteins.

Ten percent glycerol prevented the usual precipitation of human serum very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) at their isoelectric points during their preparative isoelectric focusing (IEF), IEF separated VLDL and LDL into two major fractions. The observed optical density peaks are not artifacts caused by binding of Ampholines to VLDL or LDL since no radioactivity accumulated in the fractions containing VLDL or LDL during IEF in the presence of [¹⁴C]Ampholine, and gel filtration completely separated the lipoproteins from [¹⁴C]Ampholine. These results suggest that IEF may separate subspecies of VLDL and LDL under suitable experimental conditions.     

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface. Biochemical experiments showed that both LDL and acetyl-LDL have specific receptors, the acetyl-LDL receptor being distinctly different from the LDL receptor. It is concluded that there are morphological differences in the binding of LDL and acetyl-LDL to cultured monocytes. These differences are supported by biochemical data.     

Action of lipases on low-density serum lipoproteins]

It was shown that the action of phospholipase A2 on low density serum lipoproteins (LDL) in the presence of serum albumin led to decrease in the floatation coefficient. Lipase hydrolyzed LDL triglycerides after pretreatment of the latter with phospholipase A2. Due to the action of lipases the LDL residue loses its solubility and the cholesterol-rich precipitate forms. The loss of solubility of lipoproteins treated with lipases and proteinases may occur in vivo, underlying athermao formation, i.e. can thus serve as one of the factors in the pathogenesis of atherosclerosis.     

Interaction of enzymatically modified low-density lipoproteins with fibronectin

Interaction of human low-density lipoproteins (LDL) with homologous fibronectin fixed on collagen-Sepharose was studied. LDL were digested with pepsin, the degree of hydrolysis amounting to 10%. Upon passing modified LDL through a fibronectin-collagen-Sepharose column the desorption of fibronectin occurred. Addition of the increasing amount of fibronectin to the pepsin-treated LDL solution in the presence of Ca2+ ions led to the formation of LDL-fibronectin insoluble complexes. Interaction of native LDL with fibronectin was not observed. The data suggest that enzymatic modification of LDL increasing interaction of modified LDL with fibronectin, a component of extracellular matrix, could promote the accumulation of such LDL in arterial walls.     

The interaction of prostaglandins with serum low-density lipoproteins

The interaction of human serum low-density lipoproteins (LDL) with various types of prostaglandins (PG) was studied using equilibrium dialysis, steady-state fluorescence polarization spectroscopy and photolabeling methods. Low concentrations (10(-13)-10(-9) M) of PGE1 and PGF2 alpha were shown to induce specific rearrangements of the lipids on the LDL surface, whereas the closely related PGE2 and PGF1 alpha had no effect. With fluorescent labeled LDL, the PGE1-induced changes of the steady-state fluorescence polarization (P) were shown to be time- and concentration-dependent, saturable and reversible. However, equilibrium dialysis revealed a very low binding capacity of LDL for PGE1 (approx. 1 prostaglandin molecule per 600 LDL particles). Approximately the same PGE1 concentration was sufficient to cause maximal changes of P, to enhance the binding to apolipoprotein B of a photoreactive sphingomyelin analogue inserted into the LDL surface and to alter the thermal phase behavior of the LDL surface lipids. It is proposed that the LDL surface rearrangement caused by prostaglandins is due to the interaction of prostaglandins with apolipoprotein B, resulting in formation of short-lived complexes. The mechanism of this interaction is discussed in terms of the non-equilibrium ligand-receptor interaction model proposed earlier to explain the interaction of prostaglandins with high-density lipoproteins (Bergelson, L.D. et al. (1987) Biochim. Biophys. Acta 921, 182-190). It is suggested that direct prostaglandin-lipoprotein interactions may play a role in the homeostasis of cholesterol.     

High-density lipoproteins protect endothelial cells from apoptosis induced by oxidized low-density lipoproteins

Summary Endothelial lesion by oxidized low-density liproproteins (LDL) is one of the first stages in the development of atherosclerosis. The effect of these lipoproteins can range from a functional lesion of the endothelium to death of the endothelial cells by apoptosis. High-density lipoproteins (HDL) are one of the factors which can have a protective effect against the development of atheromatous plaques. The aim of this study is to establish whether the death of endothelial cells by apoptosis induced by oxidized LDLs is prevented by HDLs. ECV304 endothelial cells and bovine aorta endothelial cells were incubated with native LDLs, oxidized LDLs, and a combination of both oxidized LDLs and HDLs. Oxidized LDLs caused a significant increase of mortality mainly by apoptosis. However, when HDLs were added together with oxidized LDLs the percentage of total mortality, the degree of lipoprotein oxidation in the medium, and the percentage of cells in apoptosis were all significantly decreased. HDLs protect against the cytotoxicity of oxidized LDLs possibly by preventing the propagation of the oxidative chain in these lipoproteins.Abbreviations LDL low-density lipoproteins - HDL high-density lipoproteins - BAEC bovine aortic endothelial cell - TBARS thiobarbituric acid-reactive substances     

Adsorption kinetics of low-density lipoproteins with Langmuir monolayer

The present work utilizes the Langmuir monolayer technique to detect the adsorption kinetics of native low-density lipoproteins and their oxidized form with the lipid monolayer. We found that low-density lipoproteins and oxidized low-density lipoproteins are able to penetrate the LM up to pressure π?=?9.9 and 11.6 mN/m. Also, the adsorption constants of both particles were found to depend strongly on the monolayer initial pressure. It is found that less compressed lipid monolayers could accommodate more native low-density lipoproteins than the oxidized ones due their higher binding affinity toward monolayers. The probable α-helical regions along the apoproteinB-100 secondary structure and average hydrophobicity could explain partially their adsorption kinetics into lipid monolayers. This simplified ‘in vitro’ study of low-density lipoprotein–monolayer interaction may serve as a step further to understand the mechanism and bioactivity of the atherosclerotic process. Also, it may shed light on the oxidized low-density lipoprotein’s role in plaque formation in the innermost arterial wall in blood vessels.