A comparison of the surface activities of human apolipoproteins A-I and A-II at the air/water interface

Surface pressure (pi) and adsorption isotherms for human apolipoproteins A-I and A-II at the air/water interface have been determined and used to deduce the probable molecular structures of the monomolecular films. The surface concentrations were measured using the surface radioactivity method to monitor the adsorption of reductively [14C]methylated apoproteins. Apolipoprotein A-I and apolipoprotein A-II are extremely surface-active proteins and adsorb to exert maximal pi values of 22 and 24 mN.m-1 respectively, at a steady-state subphase concentration of about 3.10(-5) g/100 ml (equivalent to 11 and 17 nM for apolipoprotein A-I and apolipoprotein A-II, respectively). At saturation monolayer coverage, the average molecular areas for apolipoprotein A-I and apolipoprotein A-II are 15 and 13 A2/residue, respectively. These packing densities are consistent with monolayers consisting largely of alpha-helical protein molecules lying with the long axes of the helical segments in the plane of the interface. Comparison of the molecular packings of spread and adsorbed monolayers of these proteins indicates that at low pi values, the adsorbed films are more expanded, but at high pi values, the molecular packing in both types of film is the same.     

Effect of oxidation on the properties of apolipoproteins A-I and A-II

Purified apolipoprotein A-I has been separated by reversed-phase high performance liquid chromatography (HPLC) into multiple peaks and these peaks have been characterized. One peak, apoA-Ib had a relatively longer retention time on HPLC but its retention time could be shortened by treatment by hydrogen peroxide. CNBr cleavage studies indicated that the differences in apoA-Ib and in its oxidation product, apoA-Ia, were due to the different oxidation states of methionine. This phenomenon was also observed in apoA-II, where methionine oxidation produced two more forms of this apolipoprotein in addition to the native form. These isomers were found to have different secondary structures and affinities for lipid. Model peptide analogs of the amphipathic helix with the same sequence but with methionine and methionine sulfoxide at the nonpolar face of the amphipathic helix were synthesized and studied. It was found that the lipid affinities of these synthetic peptide isomers were very different. They also differed in their secondary structures as studied by circular dichroism (CD). We propose that methionine oxidation introduces hydrophilic residues at the nonpolar face of the amphipathic helical domains of these apolipoproteins and, therefore, alters their secondary structure and lipid affinity.     

Metabolism of human apolipoproteins A-I and A-II: compartmental models

The metabolism of radioiodinated apolipoproteins (apo) A-I and A-II have been examined using the techniques of compartmental modeling. The model for apoA-I contains two plasma compartments decaying at different rates. One component of apoA-I has a residence time of 3.8 days and the second has a residence time of 6.1 days. In contrast, the apoA-II model has only one plasma component, with a residence time of 5.5 days, which decays through two distinct pathways. Twenty-seven percent of apoA-II decays through a pathway that takes 1.1 days longer to reach the urine than the remaining 73% which decays through the more direct path. These differences in the metabolism exist in both male and female populations. Comparison of fasting and nonfasting concentrations of apoA-I revealed that apoA-I concentration was elevated 0.5 standard deviations in the nonfasting samples while there was no significant difference in the apoA-II concentrations. The fasting apoA-I concentrations were found to be less stable over the study period when compared to fasting apoA-II concentrations. These findings are interpreted as indicating that apoA-I and apoA-II each have a separate metabolism which overlaps when they are present on the same lipoprotein particle. Furthermore, these findings are consistent with the concept that apoA-I metabolism is influenced more by perturbations such as dietary modulation.     

Rapid chromatographic purification of apolipoproteins A-I and A-II from human plasma

Rapid, large-scale isolation of human apolipoproteins A-I and A-II has been accomplished using two chromatographic procedures. The apolipoproteins adsorbed from plasma onto a column of phenyl-Sepharose are eluted with increasing propylene glycol concentrations. Apolipoproteins A-I and A-II can be resolved by elution with a linear 0 to 80% propylene glycol gradient. Homogeneous preparations of apo A-I and A-II are obtained following gel filtration in 3M guanidinium chloride.     

Dynamic interfacial properties of human apolipoproteins A-IV and B-17 at the air/water and oil/water interface

Viscoelastic behavior of proteins at interfaces is a critical determinant of their ability to stabilize emulsions. We therefore used air bubble surfactometry and drop volume tensiometry to examine the dynamic interfacial properties of two plasma apolipoproteins involved in chylomicron assembly: apolipoprotein A-IV and apolipoprotein B-17, a recombinant, truncated apolipoprotein B. At the air/water interface apolipoproteins A-IV and B-17 displayed wide area - tension loops with positive phase angles indicative of viscoelastic behavior, and suggesting that they undergo rate-dependent changes in surface conformation in response to changes in interfacial area. At the triolein/water interface apolipoprotein A-IV displayed maximal surface activity only at long interface ages, with an adsorption rate constant of 1.0 3 10(-)(3) sec(-)(1), whereas apolipoprotein B-17 lowered interfacial tension even at the shortest interface ages, with an adsorption rate constant of 9.3 3 10(-)(3) sec(-)(1). Apolipoprotein A-IV displayed an expanded conformation at the air/water interface and a biphasic compression isotherm, suggesting that its hydrophilic amphipathic helices move in and out of the interface in response to changes in surface pressure.We conclude that apolipoproteins A-IV and B-17 display a combination of interfacial activity and elasticity particularly suited to stabilizing the surface of expanding triglyceride-rich particles.     

Effect of storage on the measurement of apolipoproteins A-I and A-II by radial immunodiffusion

We studied the effect of storage time and conditions on the measurement of apolipoprotein A-I and A-II by radial immunodiffusion. Purified A-I and A-II standards were stable for at least 6 months before any change in immunoreactivity was detected if stored at 4 degrees C at concentrations of 0.06-0.24 mg/ml for A-I and 0.016-0.064 mg/dl for A-II in 0.84 M tetramethylurea, 6.4 M urea, and 8 mM Tris-hydrocholoride, pH 8.0. Purified A-I (0.8-1.6 mg/ml) and A-II (0.5-1.0 mg/ml) were stable for 1 year if stored at -60 degrees C in 5 mM NH4HCO3 with or without 4.2 M tetramethylurea. Serum or plasma could be stored at 4 degrees C (under conditions where evaporation and bacterial growth were minimized) for at least 46 days or at -20 degrees C for up to 3 years without any change in A-I or A-II levels. For four serum samples stored at -20 degrees C for 2 to 3 years, the coefficient of variation of measurement ranged from 6.3 to 9.8% for A-I and from 6.7 to 10.6% for A-II. Samples stored at 4 degrees C had comparable apolipoprotein levels to those stored at -20 degrees C. However, apolipoprotein levels in serum samples were 3-5% higher than those obtained on plasma samples. We conclude that purified A-I or A-II and serum and plasma can be stored for long periods without any change in the measurement of the A-I or A-II by radial immunodiffusion.     

The interfacial properties of ApoA-I and an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at the triolein/water interface

Apolipoprotein A-I (apoA-I) is the major protein in high density lipoprotein (HDL). During lipid metabolism, apoA-I moves among HDL and triacylglycerol-rich lipoproteins. The main structure and the major lipid binding motif of apoA-I is the amphipathic alpha-helix. To understand how apoA-I behaves at hydrophobic lipoprotein interfaces, the interfacial properties of apoA-I and an amphipathic alpha-helical consensus sequence peptide (CSP) were studied at the triolein/water (TO/W) interface. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-residue tandem repeat sequences that form amphipathic alpha-helices modeling the central part of apoA-I. ApoA-I or CSP added into the aqueous phase surrounding a triolein drop lowered the interfacial tension (gamma) of TO/W in a concentration- and time-dependent fashion. The gamma(TO/W) was lowered approximately 16 millinewtons (mN)/m by apoA-I at 1.4 x 10(-6) m and approximately 15 mN/m by CSP at 2.6 x 10(-6) m. At equilibrium gamma, both apoA-I and CSP desorbed from the interface when compressed and readsorbed when expanded. The maximum surface pressure CSP could withstand without being ejected (PiMAX) was 16 mN/m. The PiMAX) of apoA-I was only 14.8 mN/m, but re-adsorption kinetics suggested that only part of the apoA-I desorbed at Pi between 14.8 and 19 mN/m. However, above approximately 19 mN/m (PiOFF) the entire apoA-I molecule desorbed into the water. ApoA-I was more flexible at the TO/W interface than CSP and showed more elasticity at oscillation periods 4-128 s even at high compression, whereas CSP was elastic only at faster periods (4 and 8 s) and moderate compression. Flexibility and surface pressure-mediated desorption and re-adsorption of apoA-I probably provides lipoprotein stability during metabolic-remodeling reactions in plasma.     

The lipid transport system in the mouse, Mus musculus: isolation and characterization of apolipoproteins B, A-I, A-II, and C-III

Four of the principle apolipoproteins of murine serum have been isolated and characterized. On the basis of their physicochemical properties, they are homologous with the human and rat apoA-I, A-II, B, and C-III. The group of apolipoproteins of middle to low molecular weight, i.e., A-I, A-II and C-III, were separated from the protein moiety of high density lipoproteins (HDL) by gel filtration chromatography, followed by electrophoresis in alkaline-urea polyacrylamide gel with electrophoretic elution. Murine apoA-I, the major protein of HDL (60-80%) displayed an Mr of approximately 27,000, and was polymorphic (four prominent isoproteins with isoelectric points in the range of pH 5.5-5.7). The amino acid profiles of mouse, rat, and human apoA-I generally resembled each other, the former being distinguished by a content of one isoleucine residue per mole. Amino terminal sequence analysis revealed marked homology between the mouse, rat, dog, and human proteins; mouse and rat apoA-I differed at residues 9 and 18 with potential dissimilarities at residues 5 and 15, while the murine and canine sequences were distinct at residues 6, 9, 13, 15, and 30. Apolipoprotein A-II was a monomer, exhibiting an Mr approximately 11,000 in SDS gels; in addition, it was polymorphic (three apparent isoproteins with pI in the pH range 5.05-5.2), and resembled its human and rat counterparts in amino acid composition. ApoC-III, an acidic peptide of pI 4.74 and of Mr approximately 9,600, possessed an amino acid composition very like that of the homologous human and rat proteins. The homology of mouse apoC-III with the human protein was confirmed by NH2-terminal sequence analysis, which revealed identical amino acids in six positions (1, 2, 4, 8, 9, and 13). As shown earlier (Camus et al. 1983. J. Lipid Res. 24: 1210-1228), two forms of immunologically reacting apoB predominated in mouse VLDL and LDL. After isolation of these lipoproteins in the presence of 1 mM PMSF, the apparent sizes of the high and low Mr forms, apoBH and apoBL, were in the ranges approximately 400,000-530,000 and approximately 250,000-280,000, respectively, according to the SDS gel system. We observed that inclusion of 1 mM PMSF was essential to retard degradation of the high Mr form apoBH. The murine B proteins were isolated from apoVLDL and apoLDL by gel filtration chromatography on Sephadex G150 in anionic detergent, and displayed apparent Mr values of 460,000 (apoBH) and 250,000 (apoBL) in 3% SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies

We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.     

Hybrid association between human apolipoproteins A-I and A-II in aqueous solution and in phospholipid recombinants

The formation of hybrid association products between apolipoprotein A-I and apolipoprotein A-II from human high-density lipoprotein was investigated in solutions of these apolipoprotein and in recombinant particles with dimyristoylphosphatidylcholine (DMPC). It was found that these two proteins interact in solution to form hybrid association products, but not to a marked degree. When these two proteins were incubated together with DMPC, it was likewise found that there was little tendency to reside on the same particle, as judged from the absence of hybrid oligomers by chemical cross-linking. By a modified immunoelectrophoretic method it was found that only about 15% of the A-II and 10% of the A-I were precipitated by the heterologous antiserum; from this it is concluded that 80–90% of these proteins do not form hybrid recombinants with the other protein. These results suggest that in the delipidated state, as well as in discoidal recombinants, there do not exist strong protein-protein interactions between A-I and A-II. This implies that even in the high-density lipoprotein, where both proteins coexist in the same particle, the A-II does not stabilize the molecular structure through interactions with A-I, and its role in this molecule remains obscure.     

A comparison of two methods to investigate the metabolism of human apolipoproteins A-I and and A-II.

Two methods are compared for measuring the kinetic parameters of apolipoprotein A-I and A-II metabolism in human plasma. In the first, high density lipoprotein apoproteins were radioiodinated in situ in the lipoprotein particle (endogenous apoprotein labeling) while in the second, individually labeled apolipoprotein A-I or A-II was incorporated into the particle by in vitro incubation (exogenous apoprotein labeling). The catabolic clearance rate of exogenously labeled apolipoprotein A-I was consistently faster than that of endogenous apolipoprotein A-I. Conversely, endogenously and exogenously labeled apolipoprotein A-II were catabolized at identical rates. The fractional plasma clearance rates of endogenous apolipoproteins A-I and A-II were the same.     

Mechanism of inhibition of hepatic triglyceride lipase from human postheparin plasma by apolipoproteins A-I and A-II

The present data describe the mechanism of the inhibitory effects of human plasma apolipoproteins A-I and A-II on hydrolysis of triglyceride catalyzed by hepatic triglyceride lipase using a substrate of triolein particles stabilized with gum arabic in vitro. The experimental data could well be described by a model in which apolipoproteins bound to the surface of lipid substrate particles inhibited the enzyme reaction. The values of Km obtained were similar with or without inhibitors and the calculated saturation levels of apolipoprotein binding to the lipid were in good agreement with those obtained in independent binding experiments.     

Transport of lipid and apolipoproteins A-I and A-IV in intestinal lymph of the rat

Intestinal lipid absorption is associated with marked increases in the synthesis and secretion of apolipoprotein A-IV (apoA-IV) by the small intestine. Whether the increased intestinal apoA-IV synthesis and secretion results from increased fat uptake, increased cellular triglyceride (TG) content, or increased secretion of TG-rich lipoproteins by the enterocytes is unknown. Previous work from this laboratory has shown that a hydrophobic surfactant, Pluronic L-81 (L-81), is a potent inhibitor of intestinal formation of chylomicrons (CM), without reducing fat uptake or re-synthesis to TG. Furthermore, this inhibition can be reversed quickly by the cessation of L-81 infusion. Thus L-81 offers a unique opportunity to study the relationship between lymphatic TG, apoA-I and A-IV secretion. In this study, we studied the lymphatic transport of TG, apoA-I, and apoA-IV during both the inhibitory phase (L-81 infused together with lipid) and the subsequent unblocking phase (saline infusion). Two groups of lymph fistula rats were used, the control and the experimental rats. In the experimental rats, a phosphate-buffered taurocholate-stabilized emulsion containing 40 mumol [3H]triolein, 7.8 mumol of phosphatidylcholine, and 1 mg L-81 per 3 ml was infused at 3 ml/h for 8 h. This was then replaced by glucose-saline infusion for an additional 12 h. The control rats received the same lipid emulsion as the experimental rats, but without L-81 added, for 8 h. Lymph lipid was determined both by radioactivity and by glyceride-glycerol determination, and the apoA-I and apoA-IV concentrations were determined by rocket electroimmunophoresis assay. L-81 inhibited the rise in lymphatic lipid and apoA-IV output in the experimental rats after the beginning of lipid + L-81 infusion. Upon cessation of L-81 infusion, the mucosal lipid accumulated as a result of L-81 treatment was rapidly cleared into lymph as CM. This was associated with a marked increase in apoA-IV output; the maximal output was about 3 times that of the fasting level. There was a time lag of 4-5 h between the peak lymph lipid output and the peak lymph apoA-IV output during the unblocking phase in the experimental rats. There was also a comparable time lag between the maximal lipid and apoA-IV outputs in the control animals. Incorporation studies using [3H]leucine showed that apoA-IV synthesis was not stimulated during lipid + L-81 infusion, perhaps explaining the lack of increase in lymphatic A-IV secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coupling of photoactivatable glycolipid probes to apolipoproteins A-I and A-II in human high density lipoproteins 2 and 3

High density lipoprotein (HDL) from human serum was subfractionated into HDL2 and HDL3 by rate-zonal density gradient ultracentrifugation. The orientation of apoproteins (apo) A-I and A-II in these subfractions was investigated by use of the photosensitive glycolipid probes, 2-(4-azido-2-nitrophenoxy)-palmitoyl[1-14C]glucosamine (compound A) and 12-(4-azido-2-nitrophenoxy)-stearoyl[1-14C]glucosamine (compound B). Both probes were added to the HDL-structures in a ratio of two or three probe molecules per particle and were photoactivated by irradiation at a wavelength above 340 nm. After delipidation the probe-apoprotein adducts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the "shallow" probe (compound A) and the "depth" probe (compound B) were coupled for 10-14% (of the label added) to apoA-I and apoA-II from HDL3 and for about 6% to apoA-I and apoA-II from HDL2. By taking into account the relative amounts of apoA-I and apoA-II, it was estimated that the "shallow" probe labeled apoA-I 40% more effectively than apoA-II in both HDL2 and HDL3; the "depth" probe labeled apoA-I and apoA-II equally well in both subfractions. The data suggest that towards the surface HDL2 and HDL3 contain a relatively larger portion of apoA-I than apoA-II, whilst towards the core both subfractions are occupied by an equal portion of apoA-I and apoA-II. Application of these photolabels has failed to point out differences in the structural organization of HDL2 and HDL3.     

Polarization-modulated FTIR spectroscopy of lipid/gramicidin monolayers at the air/water interface

Monolayers of gramicidin A, pure and in mixtures with dimyristoylphosphatidylcholine (DMPC), were studied in situ at the air/H2O and air/D2O interfaces by polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Simulations of the entire set of amide I absorption modes were also performed, using complete parameter sets for different conformations based on published normal mode calculations. The structure of gramicidin A in the DMPC monolayer could clearly be assigned to a beta6.3 helix. Quantitative analysis of the amide I bands revealed that film pressures of up to 25-30 mN/m the helix tilt angle from the vertical in the pure gramicidin A layer exceeded 60 degrees. A marked dependence of the peptide orientation on the applied surface pressure was observed for the mixed lipid-peptide monolayers. At low pressure the helix lay flat on the surface, whereas at high pressures the helix was oriented almost parallel to the surface normal.     

Antibodies to the carboxyl terminus of human apolipoprotein A-I. The putative cellular binding domain of high density lipoprotein 3 and carboxyl-terminal structural homology between apolipoproteins A-I and A-II.

We have studied the binding of 125I-labeled high density lipoproteins (HDL3) to liver plasma membranes, which are thought to contain specific HDL receptor sites, using anti-peptide antibodies directed against two sites in the carboxyl-terminal region of human apoA-I. Two distinct antibody populations raised to peptides corresponding to amino acid residues 205-220 and 230-243, respectively, recognized regions of apoA-I that are exposed in the lipid environment of HDL3. However, anti-AI[230-243] IgG, but not anti-AI[205-220] IgG, recognized HDL2, suggesting that residues 205-220 of apoA-I are expressed differently in the two HDL populations. In addition, anti-AI[230-243] IgG showed strong cross-reactivity toward apoA-II. Epitope mapping studies showed that anti-AI[230-243] binds to an epitope located in the carboxyl-terminus of apoA-II, demonstrating significant structural homology between the carboxyl-terminal of apoA-II, demonstrating significant structural homology between the carboxyl-terminal regions of apoA-I and A-II, two candidate proteins for mediating the specific cellular interaction of HDL3. Fab fragments from anti-AI[205-220] and anti-AI[230-243] inhibited the binding of 125I-HDL3 to liver plasma membranes by approximately 80% and 60%, respectively. These findings are in agreement with our recent work using isolated CNBr fragments of apoA-I (Morrison, J., Fidge, N. H., and Tozuka, M. (1991) J. Biol. Chem. 266, 18780-18785), which suggest that the carboxyl-terminal region of apoA-I contains a binding domain which mediates the specific interaction of HDL3 with liver plasma membranes, possibly through the involvement of specific HDL receptors.     

Isolation of apolipoproteins A-I, A-II and E and their estimation by rocket immunoelectrophoresis in blood plasma of dis-alpha-lipoproteinemic patients

The methods for isolation of pure apolipoproteins A-I, A-II and E from the blood plasma of donors for preparation of monospecific rabbit antisera against these apolipoproteins and their estimation in human blood plasma using immunoelectrophoresis are described. It was found that the average content of apolipoprotein A-I (apo A-I) in the blood plasma of healthy males is 126.6 mg%, that of apolipoprotein A-II (apo A-II) is 56.8 mg%, that of apolipoprotein E (apo E) is 10.2 mg%. The apo A-I content in blood plasma is increased in hyper-alpha-lipoproteinemic patients and is decreased in hypo-alpha-lipoproteinemic ones, i. e. there is a direct relationship between the changes in concentration of high density lipoproteins (HDL) and apo A-I. The concentration of apo A-II in dis-alpha-lipoproteinemias varies within a narrow range. A considerable increase of the alpha-cholesterol/apo A-I ratio suggesting an increased capacity of HDL to transport cholesterol in hyper-alpha-lipoproteinemic patients is observed. There exists an indirect correlation between the changes in the contents of apo A-I and apo E in dis-alpha-lipoproteinemic patients.     

Asymmetric lipid bilayer formation stabilized by DNA at the air/water interface

The lipid bis(guanidinium)-tren-cholesterol (BGTC) is a cationic cholesterol derivative bearing guanidinium polar headgroups used for gene transfection either alone or formulated as liposomes with the zwitterionic lipid 1,2-di-[cis-9-octadecenoyl]-sn-glycero-3-phosphoethanolamine (DOPE). Previous investigations have shown its ability to strongly interact with DNA and form asymmetric lipid bilayers at the air/water interface when mixed with DOPE. Here, with a view to further investigate its physicochemical behavior, we studied the interactions of mixtures of BGTC with another zwitterionic lipid, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine, (DMPC), with DNA at the air/water interface by using the Langmuir monolayer technique coupled with Brewster Angle Microscopy (BAM) and Polarization Modulation Infra Red Reflexion Absorption (PMIRRAS) spectroscopy and we investigate DNA–BGTC/DMPC interactions. We demonstrate that when DNA is injected into the subphase in excess compared to the positive charges of BGTC, it adsorbs to BGTC/DMPC monolayers at 20 mN/m whatever the lipid monolayer composition (1/5, 2/3 or 3/2 BGTC/DMPC molar ratio) and forms an incomplete monolayer of either isotropic or anisotropic double strands depending on the BGTC content in the monolayer. Compression beyond the collapse of some mixed DNA–BGTC/DMPC (2/3 and 3/2 molar ratio) systems leads to the formation of DNA monolayers underneath asymmetric lipid bilayers characterized by a bottom layer of BGTC in contact with DNA and a top layer mainly constituted of DMPC.     

Interfacial properties of an amphipathic alpha-helix consensus peptide of exchangeable apolipoproteins at air/water and oil/water interfaces

Amphipathic alpha-helices are the main structure and the major lipid binding motif of exchangeable apolipoproteins. To understand how these apolipoproteins behave at an hydrophobic lipoprotein interface, the interfacial properties of a consensus sequence peptide (CSP) derived from three exchangeable apolipoproteins (A-I, A-IV, and E) were studied using an oil drop tensiometer at air/water (A/W) and dodecane/water (DD/W) interfaces. CSP ((PLAEELRARLRAQLEELRERLG)2-NH2) contains two 22-amino acid tandem repeat sequences that form amphipathic alpha-helices. CSP, when added into the aqueous phase, lowered the interfacial tension (gamma) of A/W and DD/W in a concentration-dependent fashion. The gammaA/W was lowered approximately 24 mn/m, and gammaDD/W approximately 31 mn/m, indicating a greater affinity of CSP for DD/W. Using the Gibbs equation for surface, the surface area per CSP molecule was estimated at approximately 702 A2 ( approximately 16 A2/amino acid) on A/W and approximately 622 A2 on DD/W ( approximately 14 A2/amino acid) suggesting that adsorbed CSP lies flat with alpha-helices in the plane of both interfaces. At equilibrium gamma, CSP desorbed from the interface when compressed and re-adsorbed when expanded. The adsorption rate was concentration-dependent, but the desorption rate was not. Less CSP desorbed from DD/W than A/W indicating that CSP has higher affinity for DD/W. Dynamic analysis of elasticity shows that the faster the oscillation period (4, 8 s) and the lower the oscillation amplitude the more elastic the surfaces. CSP can be compressed 6-12% while remaining on the surface, but large increases in pressure eject it from the surface. We suggest that surface pressure-mediated desorption and readsorption of amphipathic alpha-helices provide lipoprotein stability during remodeling reactions in plasma.