Sedimentation Properties of Dextran Sulfate-Low Density Lipoprotein Complexes

The nature of interaction between dextran sulfate and the human plasma low density lipoproteins of S_f 0–10 was investigated in high density media of glycine and glucose. The soluble complex formation between the two components was manifested by sedimentation of the lipoproteins along with dextran sulfate in the glycine and glucose media of density 1.063. The addition of sodium chloride to the mixture caused dissociation of the complex: during subsequent ultracentrifugation, flotation of lipoprotein and sedimentation of dextran sulfate occurred. However, when the complex is in the acidic glycine medium (pH 4.0), the addition of sodium chloride did not induce dissociation of the complex.

Both the solubility and the size of the complex were greatly influenced by the ratio of the two components in solution. At low relative concentrations of dextran sulfate, insoluble aggregates were formed; but the aggregates disintegrated into soluble units upon increasing the dextran sulfate concentrations. From the sedimentation patterns of dextran sulfate lipoprotein mixtures at various ratios, it was possible to estimate the ratio of the two components in the complex. In the presence of excess dextran sulfate a composite biphasic Schlieren diagram was produced as a result of the unusual Johnston-Ogston effect.     

Mechanism of heparin and serum lipoprotein interaction: effects of calcium, phosphorylcholine, and a serum fraction.

The mechanism of formation of an insoluble complex between heparin and rat serum lipoprotein has been studied. Optical density changes during the reaction, counting of the fatty acid labelled lipoproteins in the precipitates, and complexing of [14C]palmitate-labelled lipoprotein with heparin-CNBr-Sepharose were used to quantitatively determine the formation of insoluble complexes. The maximal heparin--lipoprotein complex formation requires 25--30 mM of Ca2+, but with micromolar amounts of phosphorylcholine, the reaction was saturated at only 10 mM of Ca2+. The effect of phosphorylcholine in promoting the reaction was lost when purified chylomicrons or very low density lipoproteins were used. The effect of phosphorylcholine in promoting the interaction between heparin and pure chylomicrons or very low density lipoproteins was regained when a crude serum protein factor of unwashed chylomicrons was added to the system, suggesting that rat serum contains a protein factor(s) which normally inhibits the heparin--lipoprotein interaction by raising the requirement of Ca2+. Phosphorylcholine counteracted the effect of this protein, thereby favouring the precipitation reaction in the presence of much lower concentration of Ca2+. The results have been discussed with special reference to the possibility of a relationship between mucopolysaccharides, Ca2+, lipoproteins, and arterial phospholipids in the pathogenesis of atherosclerosis.     

Interaction of Fibrinogen with Dextran Sulfate

Interactions of fibrinogen with dextran sulfate, dextran, and carboxymethyl cellulose were investigated by turbidity measurement, chemical analysis, and electrophoresis. Dextran sulfate and fibrinogen combined even in the physiological pH region where both of them have negative net charges, and formed a precipitate and soluble complex. Since no complex formation was observed in the case of dextran, it seems that the electrostatic force plays a part in complex formation. However, sodium carboxymethyl cellulose which carries -COO^- groups did not combine with fibrinogen. Therefore, it is considered that there is a specificity for the interaction among ionized groups. Further, temperature and molecular size of dextran sulfate influenced the interaction to a large extent. It is concluded from these facts that other intermolecular binding forces should be taken into consideration in addition to the electrostatic force.     

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.     

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate.

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated Kd in the 10(-4) M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.     

Utilization of free fatty acids complexed to human plasma lipoproteins by mammalian cell suspensions

The purpose of this study was to determine whether lipoprotein-bound free fatty acid could be utilized by isolated mammalian cells. Ehrlich ascites tumor cells were incubated in vitro with radioactive free fatty acids that were bound to human plasma lipoproteins. Under these conditions, lipoprotein-bound free fatty acids were readily taken up by the cells. After 2 min of incubation with free fatty acids bound to low density lipoproteins, most of the radioactivity that was associated with the cells was in the form of free fatty acids. As the incubation continued, increasing amounts of radioactivity were incorporated into CO(2) and cell lipids, particularly phospholipids. Most of the free fatty acid uptake was the result of fatty acid transfer from low density lipoproteins to the cell, not from irreversible incorporation of the intact free fatty acid-low density lipoprotein complex. Fatty acid uptake increased as the ratio of free fatty acid to low density lipoprotein was raised. When albumin was added to the medium, free fatty acid uptake decreased. A large percentage of the newly incorporated cellular radioactivity was released into the medium if the cells were exposed subsequently to a solution containing albumin. Most of the released radioactivity was in the form of free fatty acid. The results with this experimental model suggest that lipoprotein-bound free fatty acid, like albumin-bound free fatty acid, is readily available for uptake by isolated cells. The mechanism of free fatty acid utilization by the Ehrlich cell is similar when either low density lipoprotein or serum albumin serves as the fatty acid carrier.     

Mechanisms of enhancement of cholesteryl ester transfer protein activity by lipolysis

Lipoprotein lipase enhances the cholesteryl ester transfer protein (CETP)-mediated transfer of cholesteryl esters from plasma high density lipoproteins (HDL) to very low density lipoproteins (VLDL). In time course studies the stimulation of cholesteryl ester transfer by bovine milk lipase was correlated with accumulation of fatty acids in VLDL remnants. As the amount of fatty acid-poor albumin in the incubations was increased, there was decreased accumulation of fatty acids in VLDL remnants and a parallel decrease in the stimulation of cholesteryl ester transfer by lipolysis. Addition of sodium oleate to VLDL and albumin resulted in stimulation of the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. The stimulation of transfer of cholesteryl esters into previously lipolyzed VLDL was abolished by lowering the pH from 7.5 to 6.0, consistent with a role of lipoprotein ionized fatty acids. CETP-mediated cholesteryl ester transfer from HDL to VLDL was also augmented by phosholipase A2 and by a bacterial lipase which lacked phospholipase activity. When VLDL and HDL were re-isolated after a lipolysis experiment, both lipoproteins stimulated CETP activity. Postlipolysis VLDL and HDL bound much more CETP than native VLDL or HDL. Lipolysis of apoprotein-free phospholipid/triglyceride emulsions also resulted in enhanced binding of CETP to the emulsion particles. Incubation conditions which abolished the enhanced cholesteryl ester transfer into VLDL remnants reduced binding of CETP to remnants, emulsions, and HDL. In conclusion, the enhanced CETP-mediated transfer of cholesteryl esters from HDL to VLDL during lipolysis is related to the accumulation of products of lipolysis, especially fatty acids, in the lipoproteins. Lipids accumulating in VLDL remnants and HDL as a result of lipolysis may augment binding of CETP to these lipoproteins, leading to more efficient transfer of cholesteryl esters from HDL to VLDL.     

Molecular interaction between HIV-1 major envelope glycoprotein and dextran sulfate

We investigated at the molecular level the interaction between, HIV-1 recombinant gp160 (rgp160) and low-molecular-weight dextran sulfate. We demonstrate the occurrence of a specific interaction between rgp160 and sulfated dextran beads, which is saturable, pH-dependent and inhibitable by soluble dextran sulfate but not by soluble dextran. This specific interaction has a low affinity, with an estimated K_d in the 10^?4 M range. In addition, the binding of rgp160 to soluble recombinant CD4 (sT4) can only be inhibited by the preincubation of rgp160, but not of sT4, with dextran sulfate. Taken together, these results demonstrate the occurrence of a low affinity, but specific interaction between dextran sulfate and rgp160. This may account, at least in part, for the anti-HIV-1 activity of dextran sulfate.     

Effect of dextran sulfate on the interaction between very low density lipoprotein and lipoprotein lipase

The effect of dextran sulfate on the interaction between very low density lipoprotein (VLDL) and purified bovine milk lipoprotein was studied. Dextran sulfate increased VLDL-triacylglycerol hydrolysis by lipoprotein lipase about 2-fold, but did not alter the Km value for triacylglycerol in VLDL. Strong association of dextran sulfate with the VLDL-lipoprotein lipase complex was demonstrated by gel filtration on BioGel A-5m, although dextran sulfate did not bind to VLDL and only very slightly to lipoprotein lipase. These findings suggest that dextran sulfate increases triacylglycerol hydrolysis in VLDL by binding to the VLDL-lipoprotein lipase complex.     

Stimulation of cholesterol ester synthesis in macrophages by lipoproteins from normal and atherosclerotic human aorta intima

A buffer extract from homogenized human aorta was applied to a Bio-Gel A-15m column, and two cholesterol-containing peaks were resolved. Both fractions of aortic lipoproteins present in the extracts from normal and atherosclerotic intima and stimulated cholesteryl ester (CE) synthesis in J774 mouse macrophages caused unregulated loading with CE. The Vmax of CE formation in the presence of both fractions correlated with the degree of intimal atherosclerosis. An excess of both fractions did not inhibit the uptake of malondialdehyde-treated low density lipoproteins by macrophages; their interaction with the cells was not inhibited either by fucoidin or by dextran sulfate. The uptake of labeled LDL by human fibroblasts was markedly decreased with excess of both fractions. Aortic lipoprotein-mediated CE synthesis (for both fractions) was completely blocked by EDTA in fibroblasts, being decreased by 50% in macrophages.     

Interactions of high density lipoproteins with very low and low density lipoproteins during lipolysis

Interactions of high density lipoproteins (HDL) with very low (VLDL) and low (LDL) density lipoproteins were investigated during in vitro lipolysis in the presence of limited free fatty acid acceptor. Previous studies had shown that lipid products accumulating on lipoproteins under these conditions promote the formation of physical complexes between apolipoprotein B-containing particles (Biochim. Biophys. Acta, 1987. 919: 97-110). The presence of increasing concentrations of HDL or delipidated HDL progressively diminished VLDL-LDL complex formation. At the same time, association of HDL-derived apolipoprotein (apo) A-I with both VLDL and LDL could be demonstrated by autoradiography of gradient gel electrophoretic blots, immunoblotting, and apolipoprotein analyses of reisolated lipoproteins. The LDL increased in buoyancy and particle diameter, and became enriched in glycerides relative to cholesterol. Both HDL2 and HDL3 increased in particle diameter, buoyancy, and relative glyceride content, and small amounts of apoA-I appeared in newly formed particles of less than 75 A diameter. Association of apoA-I with VLDL or LDL could be reproduced by addition of lipid extracts of lipolyzed VLDL or purified free fatty acids in the absence of lipolysis, and was progressively inhibited by the presence of increasing amounts of albumin. We conclude that lipolysis products promote multiple interactions at the surface of triglyceride-rich lipoproteins undergoing lipolysis, including physical complex formation with other lipoprotein particles and transfers of lipids and apolipoproteins. These processes may facilitate remodeling of lipoproteins in the course of their intravascular metabolism.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.     

Soluble heparin proteoglycans released from stimulated mast cells induce uptake of low density lipoproteins by macrophages via scavenger receptor-mediated phagocytosis.

Stimulation of rat serosal mast cells in vitro triggers exocytosis of secretory granules from their cytoplasm. Thereupon, the granules lose their perigranular membranes, and about 40% of the heparin proteoglycans and all of the chondroitin sulfate proteoglycans that they initially contained are released into the incubation medium. At physiologic ionic strength and calcium ion concentration, the solubilized heparin proteoglycans, but not the chondroitin sulfate proteoglycans, form insoluble complexes with the low density lipoproteins (LDL) present. We calculated that the heparin proteoglycans could bind approximately seven times their own mass (Mr about 1 x 10(6)) of LDL cholesterol. Using gold-labeled LDL, we observed massive phagocytosis of the heparin proteoglycan-LDL complexes by cultured mouse macrophages in vitro, which was inhibited by cytochalasin B. Uptake of LDL by mouse macrophages was 45-fold higher in the presence of solubilized heparin proteoglycans than in their absence, and continued unabated over a 72-h period, indicating that the uptake process was not under negative feedback control. Excess amounts of acetyl-LDL or polyinosinic acid inhibited the uptake of these insoluble heparin proteoglycan-LDL complexes, indicating that their phagocytosis was mediated by scavenger receptors of the acetyl-LDL receptor type. The experiments reveal the following pathophysiologic mechanism relevant to atherogenesis: stimulated mast cells secrete soluble heparin proteoglycans capable of forming insoluble complexes with LDL and thereby trigger uptake of LDL by macrophages through scavenger receptor-mediated phagocytosis.     

Dietary saturated fatty acid content affects lymph lipoproteins: studies in the rat

We examined effects on intestinal absorption of cholesterol and triglycerides and intestinal lipoprotein formation by feeding rats diets in which saturated fatty acids (palmitic plus stearic) comprised 78%, 68%, 48%, or 38% of triglyceride fatty acids. Absorption into lymph of radiolabeled cholesterol was proportional to triglyceride absorption. The rates of absorption of these lipids were related inversely to the % saturated fatty acids fed. The distribution of newly absorbed cholesterol and triglyceride into intestinal lipoproteins differed. With increasing cholesterol absorption more was recovered in very low density lipoproteins in contrast to the appearance preferentially in chylomicrons of larger quantities of fatty acid. Lymph lipid content did not reflect a consistent pattern in relation to the experimental diet fed. The fatty acid composition of triglyceride-rich lymph lipoproteins resembled the diet closely. One-quarter of the intestinal lymph particles from rats fed the highly saturated diets was flattened and polygonal as judged by electron microscopy if cooled to room temperature; whereas with the same diets, particles collected and isolated at 37 degrees C were round. Proportions of A-I and C apolipoproteins in triglyceride-rich intestinal particles varied inversely; apoA-I increased as fat/cholesterol absorption was greater. Diet-induced alterations in plasma lipoproteins and increased circulating triglycerides in this study in rats were unrelated to the variations in intestinal absorption or lymph lipoprotein formation.     

Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.     

Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions

Procedures are described for the isolation of lipoproteins from human serum by precipitation with polyanions and divalent cations. A mixture of low and very low density lipoproteins can be prepared without ultracentrifugation by precipitation with heparin and either MnCl(2) alone or MgCl(2) plus sucrose. In both cases the precipitation is reversible, selective, and complete. The highly concentrated isolated lipoproteins are free of other plasma proteins as judged by immunological and electrophoretic methods. The low density and very low density lipoproteins can then be separated from each other by ultracentrifugation. The advantage of the method is that large amounts of lipoproteins can be prepared with only a single preparative ultracentrifugation. Polyanions other than heparin may also be used; when the precipitation of the low and very low density lipoproteins is achieved with dextran sulfate and MnCl(2), or sodium phosphotungstate and MgCl(2), the high density lipoproteins can subsequently be precipitated by increasing the concentrations of the reagents. These lipoproteins, containing small amounts of protein contaminants, are further purified by ultracentrifugation at d 1.22. With a single preparative ultracentrifugation, immunologically pure high density lipoproteins can be isolated from large volumes of serum.     

Fatty acids of glycosphingolipids from pig blood fractions

Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.     

Effects of several common long chain fatty acids on the properties and lipid composition of the very low density lipoprotein secreted by the perfused rat liver.

1. Livers from normal fed male rats were perfused in vitro with a bloodless medium which contained intially 3% bovine serum albumin and 100 mg% glucose. Albumin alone, or myristate (14 : 0), palmitate (16 : 0), palmitoleate (16 : 1), stearate (18 : 0), oleate (18 : 1), or linoleate (18:2) was infused at a constant rate (496 mumol/4 h), as a complex with albumin, during the experiment. 2. The very low density lipoprotein secreted by the liver after infusion of unsaturated fatty acids (16 : 1, 18 :1, 18 : 2) has a faster rate-zonal mobility in the ultracentrifuge and is, therefore, probably a larger particle with fewer moles of phospholipid and cholesterol relative to triacyglycerol (triacyglycerol/phospholipids/cholesterol = 100/25.1/16.4) than the very low density lipoproteins produced after infusion of saturated (14 : 0, 16 : 0, 18 : 0) fatty acids (triacyglycerol/phospholipids/cholesterol = 100/30.1/19.1). The molar ratio of phosphoipids/cholesterol of the very low density lipoprotein was similar regardless of which fatty acid was infused. The predominant fatty acid of the very low density lipoprotein or hepatic triacyglycerol, in all cases, was the infused acid. 3. We conclude that free fatty acid regulates the quantity and proportions of triacyglycerol, phospholipids, and cholesterol secreted by the liver in the very low density lipoprotein, and therefore, may secondarily influence concentrations of lipids in the very low density lipoprotein and other plasma lipoproteins circulating in vivo.     

Sedimentation Analysis of the Interacting Mixture of Dextran Sulfate and Low Density Lipoprotein

The reversible interaction between dextran sulfate (D) and the low density lipoprotein of human serum (P) was investigated by sedimentation velocity. Analysis of the velocity patterns of dextran sulfate—lipoprotein mixtures revealed that the maximum number of binding sites on dextran sulfate molecule is approximately 6. It was also shown that the species of the complex formed is affected by the mixing ratio of the two constituents: at the molar ratio (P/D) 0.69, the complex exists in average as DP_1.6 and at 0.98 as DP_2.2. The linear increment of sedimentation coefficient of the complex due to the binding of one lipoprotein molecule was 7.8S. Finally, the mechanism of precipitation of the complexes was discussed.     

Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition.

Oxidized (ox-) low density lipoproteins (LDL) is characterized by the formation of lipid peroxides and their decomposition to reactive aldehydes which covalently link to apoB in LDL. These chemical changes are believed to be responsible for the enhanced recognition of ox-LDL by receptors on macrophages in culture. When oxidation is extensive, particle aggregation also occurs. The aim of this study was to characterize aggregation formation and how this influences the interaction of ox-LDL with macrophages in culture. When LDL was oxidized by incubating at 500 micrograms of protein/ml with 10 microM Cu2+ at 20 degrees C for up to 25 h, time-dependent increases in thiobarbituric acid reactive substances, conjugated diene content, electrophoretic mobility, and fluorescence at 360 excitation/430 emission were found. Particle aggregation increased in parallel with several parameters of oxidation and increased with increasing incubation temperatures and LDL concentrations used. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apoB fragments of reproducible sizes and higher molecular weight species appeared after mild oxidation of LDL. The percent of total apoB remaining aggregated in sodium dodecyl sulfate was 50-80% at high degrees of oxidation, whereas it was far less in LDL that had been aggregated without chemical modification. This suggested that intermolecular cross-linking of apoB had occurred during oxidation of LDL at high concentrations. Degradation of ox-LDL in mouse peritoneal macrophages (MPM) increased in parallel with the degree of oxidation and with particle aggregation but reached a plateau after 12 h. Results from cross-competition studies in MPM with soluble and insoluble portions of extensively ox-LDL and with acetyl-LDL were consistent with uptake of soluble ox-LDL via both the scavenger receptor and another receptor on MPM, and uptake of the insoluble ox-LDL by an alternative mechanism.