Phospholipid/cholesteryl ester microemulsions containing unesterified cholesterol: model systems for low density lipoproteins

As models for the effects of unesterified cholesterol (UC) on the lipid organization of low density lipoprotein (LDL), microemulsions containing either egg yolk phosphatidylcholine (EYPC) or dimyristoyl phosphatidylcholine (DMPC) as the surface component, cholesteryl oleate (CO) as the core component, and varying amounts of unesterified cholesterol were prepared by sonication. Gel filtration chromatography showed coelution of each of the lipid components, demonstrating the formation of well-defined microemulsion populations. Unesterified cholesterol incorporation into the microemulsions was proportional to the composition of the original mixture at low unesterified cholesterol compositions, but reached saturation at compositions of approximately 15 and 10 mol% unesterified cholesterol for EYPC/CO and DMPC/CO microemulsions, respectively. The Stokes' radius of the microemulsions was constant and similar to native LDL for initial compositions less than 15 mol% unesterified cholesterol, but increased at compositions above 15 mol%. In both EYPC/CO/UC and DMPC/CO/UC microemulsions, no significant changes were observed for the calorimetric or Van't Hoff enthalpy for the thermal transition of the core cholesteryl ester; however, increases in the transition temperature as a function of increasing unesterified cholesterol composition suggests that unesterified cholesterol has a stabilizing effect on the core transition. In DMPC/CO/UC microemulsions, the effect of unesterified cholesterol on the surface-located DMPC could be clearly observed as a broadening of the thermal transition of the acyl chains. These results demonstrate that unesterified cholesterol is located primarily in the surface of these protein-free lipid model systems for LDL.     

Physical properties of apoprotein B in mixed micelles with sodium deoxycholate and in a vesicle with dimyristoyl phosphatidylcholine

Apoprotein B, the major apoprotein of normal human low density lipoprotein (LDL) was solubilized with sodium deoxycholate (NaDC). The protein was recombined with the phospholipid dimyristoyl phosphatidylcholine (DMPC) to produce a complex of DMPC-apoB (4:1 w/w). (Biochemistry. 22: 3170-3178. 1983). Carboxyfluorescein and [3H]dextran entrapment studies show the DMPC-apoB 4:1 (w/w) complex to encapsulate an aqueous volume of 0.17 microliter/mumol of DMPC. From the chemistry and morphology of the complex and the evidence that the complex possesses an encapsulated volume, the most appropriate structural model for this assembly is that of a phospholipid single bilayer vesicle into which apoB is incorporated. Differential scanning calorimetry (DSC) and circular dichroic spectroscopy (CD) were used to investigate the physical properties of apoB in the mixed micellar complex with NaDC and in the vesicular DMPC-apoB complex. CD studies of apoB in NaDC mixed micelles show that apoB exhibits a similar secondary structure as apoB of native LDL over the temperature range 5-30 degrees C. Reversible structural changes occur between 30 and 50 degrees C. However, above 50 degrees C, disruption of the micellar particle and endothermic protein unfolding and denaturation occur with a Tmax of 52 degrees C and an enthalpy of 0.22 cal/g apoB, as shown by DSC. The DMPC-apoB complex exhibits a reversible thermal transition centered at 24 degrees C (delta H = 3.34 Kcal/mol DMPC) which is associated with the order-disorder transition of the hydrocarbon chains of DMPC. An endothermic transition occurs over the range 53-70 degrees C (delta H = 2.09 cal/g apoB) which, as shown by CD and turbidity study, corresponds to protein unfolding-denaturation and particle disruption. CD shows that apoB in the vesicular environment undergoes a series of conformational changes. The major alterations occur over the temperature range of the order-disorder transition of the phospholipid. Between 37-60 degrees C, the conformation is similar to that observed in native LDL.     

Solubilization of low-density lipoprotein with sodium deoxycholate and recombination of apoprotein B with dimyristoylphosphatidylcholine

Apoprotein B (apoB) of human plasma low-density lipoprotein (LDL) (d 1.025-1.050 g/mL) has been solubilized with solid sodium deoxycholate (NaDC) above its critical micellar concentration. ApoB is isolated by gel-filtration chromatography as a mixed micellar complex of protein and detergent in high yield in a lipid-free form. A soluble apoB-dimyristoylphosphatidylcholine (DMPC) complex has been prepared by incubation of aqueous solutions of apoB-NaDC and DMPC-NaDC (2/1 w/w) at room temperature with detergent removal by extensive dialysis. A combination of gel chromatographic and density gradient fractionation of DMPC-apoB incubation mixtures demonstrates that a reasonably well-defined complex of DMPC and apoB is formed with a 4:1 w/w lipid:protein ratio. Negative-stain electron microscopy shows these particles to be single-bilayer phospholipid vesicles with a diameter of 210 +/- 20 A into which the apoB is incorporated. Circular dichroic spectra of NaDC-solubilized apoB show apoB to have similar conformation to that seen in the native LDL particle. However, apoB that has been complexed with DMPC exhibits more alpha-helix. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band (apparent Mr 366000) for apoB after solubilization, purification, and interaction with phospholipid. The behavior of apoB during its reassociation with phospholipid and the structural features of the DMPC-apoB particle are similar to those observed in the interaction of solubilized membrane proteins with lipid rather than that of other apo-lipoproteins.     

Orientational and motional properties of copper (II) complexes of dibenzotetraaza [14]annulenes in lipid bilayers: an ESR study.

ESR studies of two copper(II) complexes of substituted dibenzotetraaza [14]annulenes, CuL and CuLA, in dimyristoylphosphatidylcholine (DMPC) and egg yolk phosphatidylcholine (EYPC) are reported. Our data show that both complexes partition into the membranes and that the rotational motion of CuL is faster than CuLA. Analysis of the ESR spectra of these complexes in DMPC vesicles indicate that the Cu-motion parameter, which is a measure of the degree of resolution of the nitrogen hyperfine structure, changes abruptly at the main phase transition. At 1 mole %, both complexes lowered the fluid/gel phase transition temperature by 2 degrees C as measured by the Cu-motion parameter. A gradual change of the Cu-motion parameter is observed in EYPC liposomes over the same temperature range. ESR spectra of both CuL and CuLA in oriented membranes reveal that both complexes are well oriented with the plane of the complex perpendicular to the bilayer surface.     

Novel phospholipolytic activities associated with electronegative low-density lipoprotein are involved in increased self-aggregation

Electronegative low-density lipoprotein (LDL(-)) is a minor LDL subfraction present in plasma with increased platelet-activating factor acetylhydrolase (PAF-AH) activity. This activity could be involved in the proinflammatory effects of LDL(-). Our aim was to study the presence of additional phospholipolytic activities in LDL(-). Total LDL was fractionated into electropositive (LDL(+)) and LDL(-) by anion-exchange chromatography, and phospholipolytic activities were measured by fluorometric methods. Phospholipolytic activity was absent in LDL(+) whereas LDL(-) presented activity against lysophosphatidylcholine (LPC, 82.4 +/- 34.9 milliunits/mg of apoB), sphingomyelin (SM, 53.3 +/- 22.5 milliunits/mg of apoB), and phosphatidylcholine (PC, 25.7 +/- 4.3 milliunits/mg of apoB). LDL(-), but not LDL(+), presented spontaneous self-aggregation at 37 degrees C in parallel to phospholipid degradation. This was observed in the absence of lipid peroxidation and suggests the involvement of phospholipolytic activity in self-aggregation of LDL(-). Phospholipolytic activity was not due to PAF-AH, apoE, or apoC-III and was not increased in LDL(+) modified by Cu (2+) oxidation, acetylation, or secretory phospholipase A 2 (PLA 2). However, LDL(-) efficiently degraded phospholipids of lipoproteins enriched in LPC, such as oxidized LDL or PLA 2-LDL, but not native or acetylated LDL. This finding supports that LPC is the best substrate for LDL(-)-associated phospholipolytic activity. These results reveal novel properties of LDL(-) that could play a significant role in its atherogenic properties.     

Interaction of tryptic peptides of apolipoprotein B-100 with dimyristoylphosphatidylcholine

Apolipoprotein B-100, the major protein constituent of human plasma low-density lipoproteins (LDL), was carboxyamidomethylated, digested with trypsin and the water-soluble tryptic peptides were coincubated with liposomes of dimyristoylphosphatidylcholine (DMPC). At 24.3 degrees C the peptides induced lipid solubilization as evidenced by optical clearing of the lipid-peptide mixture. Lipid-peptide complexes were isolated by density-gradient ultracentrifugation in KBr and had the following properties: DMPC/peptide ratio of 5.6 (w/w); buoyant density of 1.07-1.09 g/ml; discoidal morphology (51 +/- 4 X 260 +/- 28 A) as determined by electron microscopy; and molecular weight of 1.5 X 10(6) as determined by nondenaturing polyacrylamide gel electrophoresis. Compared to liposomes and sonicated vesicles of DMPC, the lipid-peptide complexes had a more rigid structure as assessed by fluorescence polarization. Whereas intact LDL had 42% alpha-helix and 15% beta-pleated sheet, the lipid-peptide complexes contained 70% alpha-helix and less than 5% beta-pleated sheet. The lipid-peptide complexes did not bind to the fibroblast high-affinity LDL receptor. These results show that specific regions in apolipoprotein B-100 which interact with phospholipid have an amphipathic character and may represent primary sites for lipid-protein interaction in LDL.     

Effects of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron spin resonance spin labeling study.

A short pulse saturation recovery electron spin resonance technique has been used to study the effects of polar carotenoid-lutein and cholesterol on interactions of 14N:15N stearic acid spin-label pairs in fluid-phase phosphatidylcholine (PC) membranes. Bimolecular collisions for pairs consisting of various combinations of [14N]-16-, [14N]-10-, [14N]-7-, or [14N]-5-doxylstearate and [15N]-16-doxylstearate in dimyristoyl-PC (DMPC) or egg yolk PC (EYPC) membranes were measured at 27 degrees C. In the absence and presence of lutein or cholesterol for both lipid systems, the collision rates were ordered as 16:5 < 16:7 < 16:10 < 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in EYPC. Polar carotenoid-lutein reduces the collision frequency for all spin-label pairs, whereas cholesterol reduces the collision frequency for 16:5 and 16:7 pairs and increases the collision frequency in the membrane center for 16:10 and 16:16 pairs. The presence of unsaturated alkyl chains greatly reduces the effect of lutein but magnifies the effect of cholesterol in the membrane center. The observed differences in the effects of these modifiers on alkyl chain bending result from differences in the structure of cholesterol and polar carotenoid and from their different localization within the lipid bilayer membrane. These studies further confirm the occurrence of vertical fluctuations of alkyl chain ends toward the bilayer surface.     

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.     

IR spectroscopic study of phospholipid emulsions containing cholesteryl oleate

As models for the lipid organization of low density lipoproteins (LDL), protein-free aqueous emulsions are prepared from dimyristoyl phosphatidyl choline (DMPC), dipalmitoyl phosphatidyl choline (DPPC), and cholesteryl oleate (CO). Aqueous dispersions containing these lipids are sonicated and yield stable particles with diameters varying between 20 and 40 nm as measured through electron microscopy. IR spectroscopy shows that emulsions consisting of DMPC, DPPC, and CO at 3/1/1 and 1/1/1 ratios undergo specific thermal transitions, depending on their composition, that can be assigned to the phospholipids forming the surface layer of the emulsion particles and to core-located CO. However, at the 1/3/1 DMPC/DPPC/CO ratio this lipid system exhibits an order-disorder transition of the mixed phospholipids with no significant transition associated with core-located CO. Observation of the methylene C&bond;H and C&bond;D stretching modes of nondeuterated and deuterated lipids enables the packing characteristics and conformational order of each lipid to be monitored separately. The transition temperature changes compared to the temperatures for the analogous transitions in neat CO and CO-free phospholipid vesicles suggest the existence of interactions between CO and the above phospholipids in the ternary emulsion particles; these interactions are stronger at the 1/3/1 DMPC/DPPC/CO ratio. The results show that interactions between core and surface phases are dependent on the emulsion lipid composition and that these findings may be extended to native lipoproteins.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.     

Defining lipid-interacting domains in the N-terminal region of apolipoprotein B

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted alpha-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogeneous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC-dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous with lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.     

Calorimetric and spectroscopic investigation of the unfolding of human apolipoprotein B

The unfolding of human apolipoprotein B-100 in its native lipid environment, low density lipoprotein (LDL), and in a soluble, lipid-free complex with sodium deoxycholate (NaDC) has been examined using differential scanning calorimetry (DSC) and near UV circular dichroic (CD) spectroscopy. High resolution DSC shows that LDL undergoes three thermal transitions. The first is reversible and corresponds to the order-disorder transition of the core-located cholesteryl esters (CE) (Tm = 31.1 degrees C, delta H = 0.75 cal/g CE). The second, previously unreported, is reversible with heating up to 65 degrees C (Tm = 57.1 degrees C, delta H = 0.20 cal/g apoB) and coincides with a reversible change in the tertiary structure of apoB as shown by near UV-CD. No alteration in the secondary structure of apoB is observed over this temperature range. The third transition is irreversible (Tm = 73.5 degrees C, delta H = 0.99 cal/g apoB) and coincides with disruption of the LDL particle and denaturation of apoB. The ratio of delta H/delta HvH for the reversible protein-related transition suggests that this is a two-state event that correlates with a change in the overall tertiary structure of the entire apoB molecule. The second protein-related transition is complex and coincides with irreversible denaturation. ApoB solubilized in NaDC undergoes three thermal transitions. The first two are reversible (Tm = 49.7 degrees C, delta H = 1.13 cal/g apoB; Tm = 56.4 degrees C, delta H = 2.55 cal/g apoB, respectively) and coincide with alterations in both secondary and tertiary structure of apoB. The changes in secondary structure reflect an increase in random coil conformation with a concomitant decrease in beta-structure, while the change in tertiary structure suggests that the conformation of the disulfide bonds is altered. The third transition is irreversible (Tm = 66.6 degrees C, delta H = 0.54 cal/g apoB) and coincides with complete denaturation of apoB and disruption of the NaDC micelle. The ratio of delta H/delta HvH for the two reversible transitions indicates that each of these transitions is complex which may suggest that several regions or domains of apoB are involved in each thermal event.     

Cationic DMPC/DMTAP lipid bilayers: molecular dynamics study

Cationic lipid membranes are known to form compact complexes with DNA and to be effective as gene delivery agents both in vitro and in vivo. Here we employ molecular dynamics simulations for a detailed atomistic study of lipid bilayers consisting of a mixture of cationic dimyristoyltrimethylammonium propane (DMTAP) and zwitterionic dimyristoylphosphatidylcholine (DMPC). Our main objective is to examine how the composition of the DMPC/DMTAP bilayers affects their structural and electrostatic properties in the liquid-crystalline phase. By varying the mole fraction of DMTAP, we have found that the area per lipid has a pronounced nonmonotonic dependence on the DMTAP concentration, with a minimum around the point of equimolar DMPC/DMTAP mixture. We show that this behavior has an electrostatic origin and is driven by the interplay between positively charged TAP headgroups and the zwitterionic phosphatidylcholine (PC) heads. This interplay leads to considerable reorientation of PC headgroups for an increasing DMTAP concentration, and gives rise to major changes in the electrostatic properties of the lipid bilayer, including a significant increase of total dipole potential across the bilayer and prominent changes in the ordering of water in the vicinity of the membrane. Moreover, chloride counterions are bound mostly to PC nitrogens implying stronger screening of PC heads by Cl ions compared to TAP headgroups. The implications of these findings are briefly discussed.     

Composition and structure of lipopolysaccharide-human plasma low density lipoprotein complex. Analytical ultracentrifugation, 31P-NMR, ESR and fluorescence spectroscopy studies

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) prepared in vitro have been analyzed. LPS-LDL complexes were found to comprise approx. 0.24 mg LPS/mg LDL protein. The major protein of complexes was apolipoprotein apoB-100 (greater than or equal to 90-95%). Incorporation of LPS molecules into LDL was accompanied by small changes in lipid composition, i.e. the phosphatidylcholine content was diminished by approx. 11% and the free fatty acid concentration was raised 2-fold. Analytical ultracentrifugation showed that insertion of LPS into LDL results in the increase of a portion of particles with higher density (lower flotation coefficient) compared to initial LDL. As was evidenced by ESR, in LPS-LDL complexes, the phospholipid hydrocarbon chains are more ordered than in LDL. 31P-NMR spectra indicated that in LPS-LDL complexes the mobility of phospholipid polar headgroups is restricted in comparison with LDL. Application of the shift reagent (Pr3+) revealed that phospholipid molecules form a monolayer structure on the surface of complexes. Upon binding of LPS to LDL, a maximum of the apoB intrinsic fluorescence was slightly red-shifted (1-2 nm) which may testify that the localization of apoB remains nearly unchanged. For LPS-LDL complexes, the accessibility of apoB fluorophores to quenchers (I-, Cs+, acrylamide) did not dramatically differ from that of LDL. It is concluded that rather large amounts of LPS (about 9-10 molecules) can accommodate in one LDL particle without severely perturbing its original composition and structure. Moreover, in the LPS-LDL complexes, oligosaccharide chains of LPS screen notably neither phospholipid polar headgroups nor, what is very important, apoB. LPS-LDL complexes are suggested to be able in vivo to bind to cellular apoB/E receptors, possible LPS receptors and scavenger-receptors of macrophages (monocytes).     

A spin label study of the lipid boundary layer of mitochondrial NADH-ubiquinone oxidoreductase

Mitochondrial NADH-ubiquinone oxidoreductase (Complex I) is a lipoprotein enzyme containing phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin. Enzyme preparations containing endogenous cardiolipin and a range of either soyabean PC or dimyristoylphosphatidylcholine (DMPC) concentrations have been made. Using a spin-labelled fatty acid, two probe environments differing in mobility have been shown to be present. The fatty acid probe has a relative binding constant (or partition coefficient between lipid and protein) of unity. The boundary layer or lipid annulus reported by the probe has a value of approx. 300 lipid molecules per molecule of enzyme FMN in preparations containing soyabean PC, or DMPC above the phase transition temperature of the latter. In soyabean PC-replaced enzyme the apparent size of the boundary layer is independent of temperature between 30 degrees C and 14 degrees C but shows a modest increase to about 400 lipid molecules per molecule of FMN between 14 degrees C and 2 degrees C. Complex I replaced with high concentrations of DMPC gives non-linear Arrhenius plots of NADH-ubiquinone oxidoreductase activity. The results of the ESR experiments show that both boundary layer and bulk lipid must be motionally restricted for this to occur. Thus, the change in activity is probably not caused by an effect exerted directly on the catalytic activity of the enzyme but is more likely due to restriction of free diffusion of ubiquinone to its site of reduction.     

Proteolytic digestion in the elucidation of the structure of low density lipoprotein.

The apoprotein (apoB) of low density lipoprotein (LDL) is reported to be a large polypeptide, and it is proposed that there are two similar-sized subunit proteins in LDL (Smith, Dawson, and Tanford. 1972. J. Biol. Chem. 247: 3376-3381.). When apoB is isolated under conditions that minimize artifactual proteolysis, only a single, large molecular weight protein appears on polyacrylamide gel electrophoresis in SDS. To investigate the organization of apoB as it exists within native LDL, limited proteolysis with trypsin has been used as a structural probe. Tryptic digestion for 1 hr at pH 7.6 with enzyme-to-protein ratios of 1:100 and 1:5 results in the liberation of approximately 10% and 30% of apoB as smaller, water-soluble peptides. These peptides may be separated from the partially digested but still intact tryptic core (T-core) of the lipoprotein by chromatography on Sephadex G-75. Repeatedly, the 1:5 T-core of native LDL is found to contain a family of polypeptides of 14,000-100,000 molecular weight. Although they have lost significant quantities of apoprotein, these T-cores sustain an appearance of homogeneity, as studied by analytical ultracentrifugation. Their measured molecular weights do not differ appreciably from those of the native LDL, and the carbohydrate content of the 1:5 tryptic T-core of LDL is similar to that of the native LDL. In normolipemic individuals, LDL generally exists in a monodisperse state, but, in different individuals, monodisperse LDL may range in molecular weight from 2.4 to 3.9 x 10(6). Limited tryptic digestions were used to probe the organization of apoB in these different molecular weight LDL. As assayed by SDS-acrylamide gel electrophoresis of the larger polypeptides and fingerprinting of the smaller released peptides, those regions of LDL exposed to trypsin digestion are identical in monodisperse LDL of 2.5 and 3.4 x 10(6) molecular weight. Thus, the different quantities of lipid bound in these various LDL must interact with apoB so that the same regions of the apoprotein are exposed to the action of trypsin in these different molecular weight lipoproteins.     

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.     

Factors contributing to the distribution of free fatty acids among phospholipid vesicles

The distribution of free fatty acids at equilibrium after incubation of small sonicated unilamellar vesicles (SUV) with large unilamellar vesicles (LUV) of different lipid composition has been determined. Stearic acid (SA) and oleic acid (OA) showed similar preferences for SUV and LUV of egg yolk phosphatidylcholine (EYPC). Both ionized and protonated forms of the free fatty acids (FFAs) behaved similarly with respect to the equilibrium distribution between EYPC of different size. The charge of the vesicles was found, however, to be important, since both FFAs in their ionized form preferentially associated to vesicles of phosphatidylcholine (PC) as compared with vesicles of phosphatidylglycerol (PC). While SA preferred membranes in the gel state, OA showed preference for the membrane in fluid state. The insertion of both OA and SA in phosphatidylethanolamine (PE)/phosphatidylcholine vesicles is less favourable than in vesicles of pure PC. All these data suggest that membrane lipid content may play a role in determining the distribution of free fatty acids among the membranes of a cell.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance

The selectivity of interaction between bovine spinal cord myelin basic protein (MBP) and eight different spin-labeled lipid species in complexes with dimyristoylphosphatidylglycerol (DMPG) and between spin-labeled phosphatidylglycerol and spin-labeled phosphatidylcholine in complexes of MBP with various mixtures of DMPG and dimyristoylphosphatidylcholine (DMPC) has been studied by electron spin resonance (ESR) spectroscopy. In DMPC/DMPG mixtures, the protein binding gradually decreased with increasing mole fraction of DMPC in a nonlinear fashion. The lipid-protein binding assays indicated a preferential binding of the protein to phosphatidylglycerol relative to phosphatidylcholine without complete phase separation of the two lipids. The outer hyperfine splittings (2Amax) of both phosphatidylglycerol and phosphatidylcholine labeled at C-5 of the sn-2 chain (5-PGSL and 5-PCSL, respectively) were monitored in the lipid-protein complexes as a function of the mole fraction of DMPC. The increases in the value of Amax induced on binding of the protein were larger for 5-PGSL than for 5-PCSL, up to 0.25 mole fraction of DMPC. Beyond this mole fraction the spectral perturbations induced by the protein were similar for both lipid labels. The ESR spectra of phosphatidylglycerol and phosphatidylcholine labeled at C-12 of the sn-2 chain were two component in nature, indicating indicating a direct interaction of the protein with the lipid chains, at mole fractions of DMPC up to 0.25. Quantitation of the motionally restricted spin-label population by spectral subtraction again indicated a preferential interaction of the protein with phosphatidylglycerol relative to phosphatidylcholine. Up to DMPC mode fractions of 0.25, the microenvironment of the protein was enriched in DMPG.(ABSTRACT TRUNCATED AT 250 WORDS)