Human lysosomal sphingomyelinase: substrate efficacy of apolipoprotein/sphingomyelin complexes

Human apolipoprotein stimulation of sphingomyelin (SM) hydrolysis by sphingomyelinase from human skin fibroblasts has been studied. Apolipoproteins A-I, A-II, B, C-I, and E do not enhance sphingomyelin hydrolysis above control levels. In contrast, apoC-II stimulates sphingomyelin hydrolysis by approximately 2.5-fold. ApoC-III, the most potent apoprotein activator, stimulates hydrolysis by 3-4-fold. ApoC-III stimulation is not significantly different for the three different isoforms which carry 0, 1, or 2 sialic acid residues. The amino-terminal half of this apoprotein, C-III(1-40), which does not bind to phospholipid surfaces, does not activate sphingomyelinase. In contrast, the carboxyl-terminal half, C-III(41-79), which strongly binds to phospholipid surfaces, stimulates sphingomyelin hydrolysis to the same level as that produced by the intact, full-length apoprotein. Incubation of sphingomyelin vesicles with increasing proportions of apoC-III results in the formation of complexes of increasing apoC-III:SM ratio and decreasing radius. The hydrolysis of sphingomyelin in the 1:50 (mol/mol) complex was more than 2-fold greater than that of the 1:200 (mol/mol) complex. The rate of hydrolysis of egg yolk sphingomyelin in the 1:50 complex was maximal [0.9 mumol h-1 (mg of protein)-1] at the gel----liquid-crystalline phase transition temperature (Tm) of the complex (40 degrees C). The rate of hydrolysis fell markedly at either higher or lower temperature. Determination of the apparent Km and Vmax values below, at, and above Tm indicated that the temperature dependence of sphingomyelin hydrolysis was attributable primarily to changes in Vmax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of apoliprotein C-III with phosphatidylcholine vesicles. Dependence of aproprotein-phospholipid complex formation on vesicle structure.

We have studied the interaction of an apolipoprotein from human very low density lipoproteins (apoC-III) with egg yolk phosphatidylcholine in the form of single- and multi-bilayer vesicles. The reactivity of single-bilayer vesicles with apoC-III appears to be greater than that of the multi-bilayer vesicles according to several thermodynamic and spectrosconic criteria. In the complexes formed by the association of apoC-III with single-bilayer vesicles, the alpha-helical content of the peptide backbone and the apolarity of the environment around the tryptophan residues are greater than that observed in the complexes formed with the multibilayer vesicles. A higher yield and more homogeneous density distribution of lipid-apoprotein complexes results from the interaction of apoC-III with the single-bilayer vesicles relative to those obtained with the multi-bilayer vesicles. The enthalpy of association of apoC-III with phospholipid was greater for the single-shelled vesicles (25 kcal/mol apoC-III) than for the multi-shelled ones (18 kcal/mol apoC-III). The difference in reactivity of these two types of liposomes is not due to a difference in their fluidities since their fatty acid compositions are identical, but may be due to a difference in their areas of sterically accessible phospholipid, their permeabilities to the apoprotein, their radii of curvation, or a combination of these factors.     

Guanidination of notexin alters its membrane-damaging activity in response to sphingomyelin and cholesterol

To elucidate the contribution of phospholipase A₂ (PLA₂) activity of notexin to its ability to perturb membranes, comparative studies on the interaction of notexin and guanidinated notexin (Gu-notexin) with egg yolk phosphatidylcholine (EYPC), EYPC/egg yolk sphingomyelin (EYSM) and EYPC/EYSM/cholesterol vesicles were conducted. EYSM notably reduced the membrane-damaging activity of notexin against EYPC vesicles, but had an insignificant influence on that of Gu-notexin. Unlike the effects noted with notexin, inactivation of PLA₂ activity by EDTA led to a reduction in the ability of Gu-notexin to induce EYPC/EYSM vesicle leakage and to increase Gu-notexin-induced membrane permeability of EYPC/EYSM/cholesterol vesicles. The geometrical arrangement of notexin and Gu-notexin in contact with either EYPC/EYSM vesicles or EYPC/EYSM/cholesterol vesicles differed. Moreover, global conformation of notexin and Gu-notexin differed in either Ca²⁺-bound or metal-free states. These results indicate that notexin and Gu-notexin could induce membrane permeability without the involvement of PLA₂ activity, and suggest that guanidination alters the membrane-bound mode of notexin on damaging phospholipid vesicles containing sphingomyelin and cholesterol.     

Fluorescence depolarization studies and phase transition in human apoprotein . phospholipid complexes.

The microviscosity of unilamellar vesicles of dimyristoyl-3-sn-phosphatidylcholine and that of phosphatidylcholine . apoprotein complexes was followed by fluorescence depolarization after labeling with 1,6-diphenyl-1,3,5-hexatriene. The transition temperature from gel-crystalline to liquid-crystalline phase in 24 degrees C for the dimyristoyl-phosphatidylcholine vesicles and is shifted to around 30 degrees C in the complexes between phosphatidylcholine and apoA-I, apoA-II, apoC-I, apoC-III proteins while the cooperativity of the transition is decreased. At temperatures below the transition of the phospholipid, the microviscosity of the complexes of phosphatidylcholine with apoA-I, apoA-II and apoC-I proteins is lower than that of the phosphatidylcholine, while the opposite effect is observed above 30 degrees C. The phosphatidylcholine . apoprotein complexes isolated on a Sepharose 6B column have a molecular weight around 100 000 and a phosphatidylcholine/apoprotein ratio of 2--2.6 (w/w). The microviscosity measurments at 35 degrees C performed after elution of the column enable the complex to be detected. The size and microviscosity of the apoprotein . phosphatidylcholine complex is compatible with a model where the vesicular structure has disappeared and the amino acid side chains present hydrophobic interaction with the phosphatidylcholine acyl chains.     

Human high denisty apolipoprotein A-I-lysolecithin-lecithin and sphingomyelin complexes. A method for high yield recombinations to lipoprotein complexes of reproducible stoichiometry.

High denisty apolipoprotein A-1 (apoLp A-I) has been prepared in a chromatographically and immunochemically homogeneous form. This apoprotein forms trimeric and tetrameric aggregates in aqueous solutions at higher concentrations. ApoLp A-I has been recombined in almost quantitative yield in the presence of lysolecithin with phosphatidylcholine and sphingomyelin to particles of reproducible stoichiometry. Lysolecithin is not required for the interactions of lecithin and sphingomyelin with the apoprotein A-I or for the stability of these complexes. Dialysis removes most of the lysolecithin without the loss of lecithin and sphingomyelin. ApoLp A-I-lecithin particles have a molecular weight of 200 000 and contain 50 molecules lecithin and 25 of lysolecithin. ApoLp A-I-sphingomyelin complexes contain 50 sphingomyelin and 13 lysolecithin molecules. The former particles show up as discs of 100 A diameter, and the latter particles are 250 A in diameter. Their thickness was estimated as 25 A in the apoLp A-I lecithin and 60 A in the apoLp A-I-sphingomyelin particles. ApoLp A-I and lysolecithin form complexes whose densities depend on the lysolecithin concentration. Lysolecithin enhances the binding of phosphatidylcholine to apoLP A-I, yielding lipoprotein complexes with decreasing density. The yield of apoLp A-I-sphingomyelin-lysolecithin complexes is proportional to the lysolecithin concentration. The ratio of apoLp A-I to sphingomyelin in all these complexes remains constant.     

Apolipoproteins C-I, C-II, and C-III: kinetics of association with model membranes and intermembrane transfer

The apoproteins (apo) C-I, C-II, and C-III are low molecular weight amphiphilic proteins that are associated with the lipid surface of the plasma chylomicron, very low density lipoprotein (VLDL), and high-density lipoprotein (HDL) subfractions. Purified apoC-I spontaneously reassociates with VLDL, HDL, and single-bilayer vesicles (SBV) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. ApoC-I also transfers reversibly from VLDL to HDL and from VLDL and HDL to SBV. The kinetics of association of the individual apoC proteins with SBV are second order overall and first order with respect to lipid and protein concentrations. At 37 degrees C, the rates of association were 2.5 x 10(10), 4.0 x 10(10) and 3.8 x 10(10) M-1 s-1 for apoC-I, apoC-II, and apoC-III, respectively. Arrhenius plots of association rate vs temperature were linear and yielded activation energies of 11.0 (apoC-I), 9.0 (apoC-II), and 10.6 kcal/mol (apoC-III). The kinetics of vesicle to vesicle apoprotein transfer are biexponential for intermembrane transfer, indicating two concurrent transfer processes. Rate constants at 37 degrees C for the fast component of dissociation were 11.7, 9.5, and 9.9 s-1, while rate constants for the slow component were 1.3, 0.6, and 0.9 s-1 for apoC-I, apoC-II, and apoC-III, respectively. The dissociation constants, Kd, of apoC-I, apoC-II, and apoC-III bound to the surface monolayer of phospholipid-coated latex beads were 0.5, 1.4, and 0.5 microM, respectively. These studies show that the apoC proteins are in dynamic equilibrium among phospholipid surfaces on a time scale that is rapid compared to lipolysis, lipid transfer, and lipoprotein turnover.     

Ether phosphatidylcholines: comparison of miscibility with ester phosphatidylcholines and sphingomyelin, vesicle fusion, and association with apolipoprotein A-I

Nonhydrolyzable matrices of ether-linked phosphatidylcholines (PCs) and sphingomyelin have been used to study the mechanism of action of lipolytic enzymes. Since ether PCs, sphingomyelin, and ester PCs vary in the number of hydrogen bond donors and acceptors in the carbonyl region of the bilayer, we have examined several physical properties of ether PCs and sphingomyelin in model systems to validate their suitability as nonhydrolyzable lipid matrices. The intermolecular interactions of ether PCs with ester PCs, sphingomyelin, and cholesterol were investigated by differential scanning calorimetry. Phase diagrams constructed from the temperature dependence of the gel to liquid-crystalline phase transition of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether) and 1,2-O-ditetradecyl-sn-glycero-3-phosphocholine (DMPC-ether) with both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) demonstrated complete lipid miscibility in the gel and liquid-crystalline phases. Additionally, phase diagrams of egg yolk sphingomyelin (EYSM) with DMPC or DMPC-ether and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-O-dioctadecyl-sn-glycero-3-phosphocholine (DSPC-ether) demonstrated no major differences in miscibility of EYSM in ester and ether PCs. The effect of 10 mol % cholesterol on the thermal transitions of mixtures of ester and ether PCs also indicates little preference of cholesterol for either lipid. The fusion of small single bilayer vesicles of DMPC, DMPC-ether, DPPC, and DPPC-ether to larger aggregates as determined by gel filtration indicated that the ester PC vesicles were somewhat more stable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Displacement of the human apoprotein A-I by the human apoprotein A-II from complexes of (apoprotein A-I)-phosphatidylcholine-cholesterol

Reassembly experiments, involving isolated human apoproteins A-I and A-II and (dimyristoylglycerophosphocholine)-cholesterol vesicles were performed with apoprotein mixtures at apoprotein A-I/A-II molar ratios varying between 0 and 3. The apoproteins were incubated at 24 degrees C. 28 degrees C and 32 degrees C with either pure dimyristoyl-glycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 2, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after labeling the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A-I and A-II were quantified separately by immunonephelometry. The content of apoprotein A-I was also monitored by measuring the intrinsic tryptophan fluorescence. The results suggest that apoprotein A-II has a greater affinity than apoprotein A-I for the phospholipid-cholesterol vesicles and that apoprotein A-II is able to quantitatively displace apoprotein A-I from the lipid-protein complexes. The content of apoprotein A-II in the complexes increases proportionally to the concentration of apoprotein A-II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A-II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A-I is displaced by 2 mol human apoprotein A-II.     

Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles.

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.     

Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase

The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.     

Monomeric state and Ca2+ transport by sarcoplasmic reticulum Ca2(+)-ATPase, reconstituted with an excess of phospholipid

The structural basis for Ca2+ transport was examined in vesicles reconstituted with an excess of phospholipid by a cholate dialysis procedure. Unincorporated protein and vesicles with a relatively high protein content were removed by sucrose density centrifugation (3-12%), leaving a fraction of lipid-rich vesicles (lipid to protein weight ratio 800-900:1) with a high coupling ratio (1.0) and transport capacity (25 mumol/mg protein, after Ca-phosphate loading). Freeze-fracture analysis showed that the reconstituted vesicles had a remarkably narrow size distribution (diameter 794 +/- 77 A (S.D.], suitable for stereological analysis. Intramembranous particles were dispersed and occurred with a low frequency in the fractured shells, also before sucrose fractionation. It was calculated that the number of intramembranous particles corresponded to the number of Ca2(+)-ATPase polypeptide/vesicle. A ratio of unity between particles and polypeptide chains was also obtained from the density of particle distribution on flat surfaces of fused vesicles, prepared by sucrose fractionation. The size of the particles formed a broad distribution, having a peak value around 60-67 A, both in the reconstituted preparation and sarcoplasmic reticulum vesicles. No evidence for protein-protein interactions was found in chemical cross-linking experiments. It is concluded that the intramembranous particles in the reconstituted preparations are referable to monomeric Ca2(+)-ATPase which is capable of transporting Ca2+ inside the vesicles. The implications of the observations for the associational state of Ca2(+)-ATPase at high protein concentration are considered in relation to previous ultrastructural investigations of membranous Ca2(+)-ATPase in native and two-dimensional-crystalline forms.     

Different interactions of egg-yolk phosphatidylcholine and sphingomyelin with detergent bile salts

To examine physical-chemical aspects of bile salt-phospholipid interactions that could contribute to preferential phosphatidylcholine (PC) secretion into bile, we have compared transitions between vesicles and micelles in model systems containing taurocholate (TC) and either egg-yolk PC (EYPC), egg-yolk sphingomyelin (EYSM), buttermilk SM (BMSM) or dipalmitoyl PC (DPPC). Phase transitions from micelles to vesicles were observed at 4-fold dilution of serially diluted EYPC/TC systems, but not earlier than at 16-fold dilution of SM/TC or DPPC/TC systems, indicating lower concentrations of the detergent required for micellization in the case of SM or DPPC. Cryo-transmission electron microscopy of phase transitions initiated by addition of TC to phospholipid vesicles revealed extremely long SM-containing intermediate structures, but shorter EYPC-containing intermediate structures. Again, larger amounts of bile salt were required to induce phase transitions in the case of EYPC compared to SM. Sizes of TC-phospholipid micelles increased progressively upon increasing phospholipid contents in the rank order: DPPC-TC相似文献   

Incorporation of cholesterol into high density lipoprotein recombinants.

Apo-A-1, the principal apoprotein of high density lipoprotein, was incubated with cholesterol containing liposomes of dimyristoyl lecithin, lecithin from high density lipoprotein or sphingomyelin. Conditions were chosen to give 100% conversion of cholesterol-free liposomes into recombinants which were isolated by density gradient ultracentrifugation. For all phospholipids, there was a progressive decrease in incorporation of lipid into recombinants with increasing cholesterol/phospholipid ratio. The cholesterol/phospholipid ratio of recombinants was ～ 45% of unreacted liposomes, for all initial cholesterol/phospholipid ratios. The reduced cholesterol content suggests exclusion of cholesterol from a fraction of recombinant phospholipid, probably a boundary layer in contact with apo A-1.     

The interaction of apolipoprotein A-I with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine

The interaction between apolipoprotein A-I and small unilamellar vesicles of dipalmitoylphosphatidylcholine at the lipid phase transition resulted in complete release of vesicle contents at molar ratios of lipid to protein from 4000:1 down to 50:1. This indicated the existence of two types of stable complexes: a vesicular apo-A-I complex with a maximum of two to three apo-A-Is/vesicle, and a micellar complex (disc) with a stoichiometry of about 50 phosphatidylcholines/apo-A-I (mol/mol). We characterized the complexes by density gradient centrifugation, by gel filtration, and by immunoprecipitation using an anti-apo-A-I antibody. The morphology of the discs was similar to that of previously reported discs. Apo-A-I-induced release of vesicle contents was monitored by the relief of self-quenching of vesicle-encapsulated carboxyfluorescein. Using this assay we characterized the nature of the interaction between apo-A-I and phospholipid vesicles. The formation of complexes between vesicles and apo-A-I followed a two-step process; below or above the lipid phase transition temperature (Tc), apo-A-I bound to phosphatidylcholine vesicles but caused little leakage of contents. Kinetic analysis of the interaction between apo-A-I and dipalmitoylphosphatidylcholine vesicles below Tc indicated that about 1 in 500 collisions leads to a stable apo-A-I-vesicle complex. The second step involved passage of those complexes through Tc, which resulted in a very rapid transition into discs or vesicular complexes. Vesicular complexes contain apo-A-I which was no longer capable of interacting with pure lipid. Discs, on the other hand, interacted with vesicles at their phase transition.     

Apolipoprotein and lipid distribution between vesicles and HDL-like particles formed during lipolysis of human very low density lipoproteins by perfused rat heart

A study was undertaken to determine the relative association of lipid and apolipoproteins among lipoproteins produced during lipolysis of very low density lipoproteins (VLDL) in perfused rat heart. Human VLDL was perfused through beating rat hearts along with various combinations of albumin (0.5%), HDL2, the infranatant of d greater than 1.08 g/ml of serum, and labeled sucrose. The products were resolved by gel filtration, ultracentrifugation, and hydroxylapatite chromatography. The composition of the lipoprotein products was assessed by analysis of total lipid profiles by gas-liquid chromatography and immunoassay of apolipoproteins. A vesicle particle, which trapped and retained 1-2% of medium sucrose, co-isolated with VLDL and VLDL remnants by gel filtration chromatography but primarily with the low density lipoprotein (LDL) fraction when isolated by ultracentrifugation. The vesicle was resolved from apoB-containing LDL lipolysis products by hydroxylapatite chromatography of the lipoproteins. The vesicle lipoprotein contained unesterified cholesterol (34%), phosphatidylcholine and sphingomyelin (50%), cholesteryl ester (6%), triacylglycerol (5%), and apolipoprotein (5%). The apolipoprotein consisted of apoC-II (7%), apoC-III (93%), and trace amounts of apoE (1%). When viewed by electron microscopy the vesicles appeared as rouleaux structures with a diameter of 453 A, and a periodicity of 51.7 A. The mass represented by the vesicle particle in terms of the initial amount in VLDL was: cholesterol (5%), phosphatidylcholine and sphingomyelin (3%), apoC-II (0.5%), apoC-III (2.2%). The majority of the apoC and E released from apoB-containing lipoproteins was associated with neutral-lipid core lipoproteins proteins which possessed size characteristics of HDL. The vesicles were also formed in the presence of HDL and serum and were not disrupted by serum HDL. It is concluded that lipolysis of VLDL in vitro results in the production of VLDL remnants and LDL apoB-containing lipoproteins, as well as HDL-like lipoproteins. A vesicular lipoprotein which has many characteristics of lipoprotein X found in cholestasis, lecithin: cholesterol acyltransferase deficiency, and during Intralipid infusion is also formed. The majority of apolipoprotein C and E released from apoB-containing lipoproteins is associated with the HDL-like lipoprotein. It is suggested that the formation and stability of the vesicle lipoprotein may be related to the high ratio of cholesterol/phospholipid in this particle.     

The surface lipid layer of human low density lipoprotein probed by dipyrenyl phospholipids

Properties of the surface lipid-protein layer of human low density lipoprotein (LDL) have been studied with fluorescent phosphatidylcholine analogues containing a pyrenyl fatty acid of variable length at both sn-1 and sn-2 position of the glycerol moiety. Only intramolecular excimer formation takes place at low concentrations, as indicated by the independence of the ratio of excimer to monomer fluorescence intensities (E/M) on the amount of the incorporated dipyrenyl phospholipid. The E/M parameter which depends on the fluidity of the probe's environment were measured for a series of dipyrenyl phospholipids in three systems, i.e. in LDL, LDL-like lipid particles (LDp) and small unilamellar phosphatidylcholine/sphingomyelin/cholesterol vesicles (SUV). The data indicate that the fluidity of the phospholipid acyl chain region decreases in the order: SUV greater than LDp greater than LDL. This suggests that interactions with both the core lipids and the protein moiety (apoB-100) contribute to the rigidity of the surface lipid layer of LDL. Dipyrenyl phospholipids also detect the thermotropic transition of the core lipids of both LDL and LDp, suggesting that this transition influences the fluidity of the surface lipid layer.     

Effect of different neutral phospholipids on apolipoprotein binding by artificial lipid particles in vivo

Soybean triacylglycerol particles, stabilized with egg yolk sphingomyelin (SPH), phosphatidylcholine (PC), phosphatidylethanolamine (PE), or PC-PE mixtures, with diameters ranging from 170 to 550 nm were prepared by sonication and isolated by ultracentrifugation. Binding of apoproteins to the lipid particles was studied in vivo using the strategy of Connelly and Kuksis. The recoveries of the injected particles, which had decreased in size and undergone minimal changes in lipid composition, ranged from 70% and 57% for SPH- and PC-stabilized particles to 14% for particles stabilized with egg yolk PC-PE mixtures. The apoprotein (apo) composition of the recovered particles showed qualitative and quantitative differences, which were affected by the number of washes during isolation. After four washes, the SPH and PC particles contained apoE, apoC-II, and apoC-III as major components and apoA-IV as minor components. In addition, all particles, except those stabilized with egg yolk PC, contained large amounts of albumin. In contrast to egg yolk PC, the dipalmitoyl PC particles bound albumin as a major component. The recovered PC-PE and PE particles were characterized by a relative decrease of apoC and greatly increased binding of albumin. The higher rate of clearance of the PE-containing particles was attributed to a relative absence of apoC-III, which is known to delay hepatic uptake of lipid particles containing it, and to a more rapid hydrolysis of PE by lipoprotein lipases. Since PE occurs as a minor and variable component of chylomicrons and plasma lipoproteins, the present observations are of physiological interest.     

Relationships between membrane lipid composition and biological properties of rat myocytes. Effects of aging and manipulation of lipid composition

Cultures of newborn rat heart myocytes undergo major age-related alterations as demonstrated by comparing 5-6-day-old cells ("young cells") and 14-15-day-old cells ("old cells"). This includes: changes from spherical to elongated shape; sphingomyelin and cholesterol level/cell increase by 100% and 50%, respectively, while the phosphatidylcholine is reduced by 15-20% with almost no change in content of total phospholipids. There is a 50% increase in total protein content/cell while DNA content remain constant. The specific activity of seven marker enzymes representing most subcellular organelles is increased. Beating rate is reduced from 160 +/- 20 to 20 +/- 20 beats min-1. All the above age-dependent alterations are affected by modification of cellular polar lipid composition. Small unilamellar vesicles of egg phosphatidylcholine added to the growth medium of old cells serve as donor of egg phosphatidylcholine to the cells and as acceptor of cellular sphingomyelin and cholesterol. Sphingomyelin-phospholipid exchange can be separated from cholesterol depletion either by using vesicles of egg phosphatidylcholine/cholesterol mixtures which serve only in the phospholipid exchange process, or by small unilamellar vesicles of sphingomyelin which act only as efficient cholesterol acceptors. Such experiments indicated that the major response of old cells is to alteration in the phosphatidylcholine to sphingomyelin mole ratio, while changes in the cholesterol level induce smaller effects. Thus, reversal of phosphatidylcholine to sphingomyelin mole ratio to the values shown by young cells reverse cellular functions and features which were altered by cell aging to levels found in young cells. This includes: increase in the beating rate back to 160 +/- 20, reduction in the total protein level and in the specific activity per DNA content of seven marker enzymes and reappearance of spherical cell shape. These results suggest that membrane lipid composition has major influence on cellular properties which as described in the accompanying paper (Yechiel, E., Barenholz, Y., and Henis, Y. I. (1985) J. Biol. Chem. 260, 9132-9136), may be mediated through the organization and dynamics of the cell membranes.     

Reconstitution of apolipoprotein A-I from human high density lipoprotein with bovine brain sphingomyelin

Reconstitution of apolipoprotein A-I was found to occur readily with bovine brain sphingomyelin (BBSM), with a maximum rate occurring at a temperature of 28 degrees C, a temperature approximating the phase transition temperature for this naturally occurring phospholipid. At BBSM:A-I weight ratios of 7.5:1 or less, a single recombinant product was observed which contained three A-I molecules per particle, which had a BBSM:A-I molar ratio of 360 to 1 and which appeared in the electron microscope as a discoidal complex with a thickness of 68 A and a diameter of 217 A. By these criteria, as well as by gel filtration, this product appears very similar to that obtained by recombination of A-I with phosphatidylcholine at elevated ratios of phospholipid/protein. No evidence was found for the existence of any BBSM:A-I complexes comparable to the smaller lecithin:A-I complex containing 200-250 mol of phospholipid and two A-I molecules per complex which has been previously reported. At BBSM:A-I ratios of 15:1 (w/w), a new type of complex was observed which was discoidal by electron microscopy but possessed a larger diameter (390 A) and higher phospholipid:protein molar ratio (535:1) than has been observed previously for recombinant complexes. The BBSM:A-I complexes were found to be significantly more resistant to denaturation by guanidine hydrochloride than the dimyristoyl phosphatidylcholine:A-I recombinant complexes. It is concluded that the mechanisms of interaction between apolipoprotein A-I and either bovine brain sphingomyelin or phosphatidylcholines are similar, but that the nature of the protein-lipid interactions with BBSM are such as to produce larger and more stable complexes than are observed with the phosphatidylcholines.     

The interaction of liposomes containing intrinsic erythrocyte membrane proteins with lipid monolayers at air/water and oil/water interfaces

The main intrinsic membrane proteins of the human erythrocyte membrane, glycophorin and the anion transporter, were isolated by extraction with Triton X-100 and ion-exchange chromatography. After removal of detergent the extract consisted of proteolipid vesicles with a lipid:protein molar ratio in the range 50-60 and a diameter of the order of 200 nm. The interaction between these vesicles and dipalmitoylphosphatidylcholine (DPPC), cholesterol and cholesterol:DPPC (2:1 molar ratio) monolayers at air/water and n-decane/water interfaces has been studied. The vesicles interact with the monolayers, rapidly causing large increases in surface pressure. Limiting values of surface pressure, 39.4-43 mN . m-1 at air/water and 31.5-33.4 mN . m-1 at the n-decane/water interface, were reached at protein levels above 1 microgram . ml-1. At the air/water interface, and probably at the n-decane/water, surface pressure increases were limited by monolayer collapse. Compression isotherms and surface potential measurements indicated that material from the proteolipid vesicles entered the monolayer phase. In contrast to proteolipid vesicles, injection of protein-free liposomes beneath the monolayer resulted in smaller, slower increases in surface pressure. Thus, the presence of intrinsic membrane proteins in vesicles greatly facilitated the transfer of material into the lipid monolayer.