Studies of the lipid binding characteristics of the apolipoproteins from human high density lipoprotein. II. Calorimetry of the binding of apo AI and apo AII with phospholipids.

The interactions of lysophosphatidylcholine and synthetic 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC) liposomes with the isolated HDL-apolipoproteins, apo AI and apo AII, has been studied by microcalorimetry. Complex formation is a highly exothermal process characterized by a maximal enthalpy of about --200 kcal/mol of apoprotein when added to DMPC at 28 degrees C in 0.05 M sodium carbonate/bicarbonate buffer, pH 9.6. For the apo AI apoprotein, the binding consists of two processes, one endothermal occurring at low phospholipid/protein ratios and one exothermal predominant at higher phospholipid levels. The endothermal process has been attributed to a lipid-induced disaggregation of the apo AI while the exothermal process is similar to the binding of apo AII or apo HDL to phospholipids. The binding of a constant AI and apo AII, demonstrates the existence of a maximal association at a 1 : 1 molar ratio of the apolipoproteins. The sequential binding of DMPC to apo AI and apo AII suggests the existence of cooperativity between the two apoproteins in phospholipid binding as apo AII promotes the incorporation of apo AI into a protein-phospholipid complex.     

Human apolipoprotein A-I forms thermally stable complexes with anionic but not with zwitterionic phospholipids

The mechanism of the association of human plasma apolipoprotein A-I (apo A-I) with the acidic phospholipids, dimyristoylphosphatidylglycerol (DMPG), egg yolk phosphatidylglycerol, and dioleoylphosphatidylserine as well as with the zwitterionic dimyristoylphosphatidylcholine (DMPC) has been studied using turbidimetry, circular dichroism, high-sensitivity differential scanning calorimetry, and electron microscopy. The association of apo A-I with multilamellar liposomes of acidic phospholipids is rapid over a broad temperature range at and above the temperature of the lipid gel to liquid crystalline transition, Tc. This is in contrast to zwitterionic phosphatidylcholine which recombines with apo A-I only over a narrow temperature range around Tc. The complex of apo A-I with DMPC denatures at elevated temperatures giving rise to a calorimetrically detectable transition. The temperature range and width of this transition is shown to be markedly dependent on the heating rate. This is again in contrast to apo A-I recombinants with DMPG which show no calorimetrically detectable thermal denaturation, at least in a temperature range up to 100 degrees C. Also circular dichroism data indicate high resistance of apo A-I to thermal unfolding in the presence of DMPG. It is concluded that the complexes of apo A-I with DMPC are thermodynamically stable only at temperatures near Tc, whereas above and below this temperature range the stability of these recombinants is determined by kinetic factors. In contrast, complexes of apo A-I with DMPG and other acidic phospholipids may be thermodynamically stable over a wide temperature range greater than or equal to Tc. In spite of these fundamental differences between zwitterionic and acidic phospholipids in their mode of association with apo A-I, the binding affinity and the morphology of the recombinants are similar. Both apo A-I X DMPC and apo A-I X DMPG complexes form lipoprotein particles having a discoidal shape.     

Expression of the human serum apolipoprotein AI and AII genes in Xenopus laevis oocytes. Lipid-associated secretion of gene products

The two major apolipoproteins of plasma high-density lipoproteins (HDL) are apolipoprotein AI (apo AI) and AII (apo AII). The apo AI and the correctly oriented apo CIII genes separated by 2.6 kb were obtained by fusion of two human lambda-genomic clones. The apo AII gene was isolated as a 3 kb clone. These apolipoprotein genes have been injected independently and together into Xenopus laevis oocytes and their expression studied. Both apolipoprotein genes were transcribed and translated into their preproforms and processed in Xenopus laevis oocytes to their proforms. They were secreted into the medium associated with newly synthesized phospholipids and neutral lipids as particles floating in the high-density lipoprotein range between 1.12 and 1.21 g/ml. Secreted apo AI is associated mainly with newly synthesized phosphatidylethanolamine and little triglyceride, apo AII with phosphatidylethanolamine, lysophosphatidylethanolamine and neutral lipids. Simultaneous injection of the apo AI and apo AII genes led to the secretion of both apoproteins which separated into two bands during CsCl-density gradient centrifugation. The heavier particles were associated with proapo AI and AII, phosphatidylethanolamine (greater than 90%) and traces of lysophosphatidylethanolamine as lipid components. Proapo AII was immunoprecipitated from the less dense fraction and found to be mainly associated with lysophosphatidylethanolamine. Radiolabelled newly synthesized apolipoproteins in secreted particles were characterized by immunoprecipitation after delipidation of the secreted lipoprotein particles. The oocyte-system proved very suitable for studies of the expression of serum apolipoprotein genes, the assembly of the apolipoproteins with specific lipids to lipoprotein particles and their secretion.     

Interaction between high-density lipoprotein subpopulations in apo B-free and abetalipoproteinemic plasma.

Two populations of high-density lipoprotein (HDL) particles exist in human plasma. Both contain apolipoprotein (apo) A-I, but only one contains apo A-II: Lp(AI w AII) and Lp(AI w/o AII). To study the extent of interaction between these particles, apo B-free plasma prepared by the selective removal of apo B-containing lipoproteins (LpB) from the plasma of three normolipidemic (NL) subjects and whole plasma from two patients with abetalipoproteinemia (ABL) were incubated at 37 degrees C for 24 h. Apo B-free plasma samples were used to avoid lipid-exchange between HDL and LpB. Lp(AI w AII) and Lp(AI w/o AII) were isolated from each apo B-free plasma sample before and after incubation and their protein and lipid contents quantified. Before incubation, ABL plasma had reduced levels of Lp(AI w AII) and Lp(AI w/o AII), (40% and 70% of normals, respectively). Compared to the HDL of apo B-free NL plasma, ABL HDL had higher relative contents of free cholesterol, phospholipid and total lipid, and contained more particles with apparent hydrated Stokes diameter in the 9.2-17.0 nm region. These differences were particularly pronounced in particles without apo A-II. Despite their differences, the total cholesterol contents of Lp(AI w AII) increased, while that of Lp(AI w/o AII) decreased in all five plasma samples and the amount of apo A-I in Lp(AI w AII) increased by 6-8 mg/dl in four during the incubation. These compositional changes were accompanied by a relative reduction of particles in the 7.0-8.2 nm Stokes diameter size region and an increase of particles in the 9.2-11.2 nm region. These data are consistent with intravascular modulation between HDL particles with and without apo A-II. The observed increase in apo A-II-associated cholesterol and apo A-I, could involve either the transfer of cholesterol and apo A-I from particles without apo A-II to those with A-II, or the transfer of apo A-II from Lp(AI w AII) to Lp(AI w/o AII). The exact mechanism and direction of the transfer remain to be determined.     

DNA polymorphisms of the apolipoprotein AII and AI-CIII-AIV genes: a study in men selected for differences in high-density-lipoprotein cholesterol concentration.

We have investigated the frequencies of RFLPs of the apolipoprotein (apo) AII gene and of the apo AI-CIII-AIV gene cluster in 109 men, selected from a random sample of 1,910 men aged 45-59 years, to cover a wide range of plasma high-density-lipoprotein (HDL)-cholesterol concentration. There was no significant difference in apo AI or apo AII RFLP allele frequency between groups of individuals with high and low HDL-cholesterol concentration. However, the apo AI PstI RFLP showed an association with genetic variation determining the plasma concentration of apo AI in this sample. Genetic variation in the apo AI-CIII-AIV gene region, as defined by haplotypes, accounted for 16% of the phenotypic variance in the apo AI concentration and for 8% of the phenotypic variance in HDL-cholesterol concentration. There was no significant association between alleles of the apo AII MspI RFLP and genetic variation determining apo AII or HDL concentration. The data demonstrate that genetic variation in the apo AI-CIII-AIV gene cluster is involved in determining the serum concentration of apo AI in this sample of clinically well individuals.     

Molecular modeling of the amphipathic helices of the plasma apolipoproteins.

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yielded a three-dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid-protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins.     

The interaction of purified acetylcholinesterase from pig brain with liposomes.

The binding of pig brain acetylcholinesterase to artificial phospholipid membranes was investigated at different temperatures. Calculation of the thermodynamic parameters revealed a small negative enthalpy change, but a large negative change in the free energy and a large positive change in the entropy on binding. The large entropy change might be interpreted as being responsible for forming the enzyme-membrane complex and was indicative of hydrophobic interactions between lipid and protein. This conclusion would also favour the hypothesis that the enzyme was an integral protein. Further support for this theory was provided by the study of acetylcholinesterase binding to liposomes containing the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Lowering the temperature below the transition temperature or incorporating cholesterol into the liposomes decreased enzyme binding. Both factors could be interpreted as decreasing the fluidity of the hydrocarbon side chains of the phospholipids, causing an increase in bilayer thickness due to closer packing of side chains. This membrane condensation would certainly not favour the binding of integral protein molecules.     

Lipid and apolipoprotein levels in six Solomon Island societies differ from those in a U.S. white population

Levels of plasma cholesterol, triglycerides, and apolipoproteins (apo) AI, AII, and E in 560 males and 744 females from six Solomon Island societies were compared with levels in age- and sex-matched participants in the Rochester Family Heart Study (RFHS). The overall average cholesterol, triglyceride, apo AI, and apo AII levels for all the Solomon Island societies were significantly lower than levels for the RFHS (P less than 0.001). The mean level of apo E for these societies was significantly higher than levels in RFHS in spite of the fact that the levels of triglycerides were significantly lower. Normally, apo E is a major constituent of triglyceride-rich very-low-density lipoprotein (VLDL). For both sexes, none of the Solomon Island societies showed a significant correlation of plasma cholesterol levels with apo E. In the RFHS, this correlation was 0.50 in males and 0.43 in females. Mean apo E levels are estimated to be 4.15-6.0% of the high-density lipoprotein (HDL) protein in the different Solomon Island societies. This study establishes a distinctive Solomon Island lipid profile characterized by the high apo E levels, which appear to be associated primarily with the HDL particle, whereas, in normal Western populations, it is associated primarily with VLDL, and only small quantities are associated with HDL.     

Fourier-transform infrared studies of CaATPase/phospholipid interaction: survey of lipid classes

CaATPase from rabbit skeletal muscle has been isolated, purified, delipidated, and reconstituted with retention of ATPase activity into lipid vesicles consisting respectively of 1,2-dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), and egg sphingomyelin. The effect of the enzyme on phospholipid order and melting characteristics were determined with Fourier-transform infrared spectroscopy. Taken together with prior data from this laboratory for 1,2-dipalmitoylphosphatidylcholine and 1,2-dioleoylphosphatidylcholine (DOPC), as well as for native sarcoplasmic reticulum (SR), three types of lipid response to protein incorporation have been observed: (1) Phospholipids with high levels of acyl chain unsaturation (DOPC or native SR) have their lipid acyl chains slightly ordered by CaATPase incorporation. The effect of protein on the gel-liquid crystal phase transition cannot be easily determined, since the cooperative melting even in these systems occurs at temperature well below 0 degrees C. (2) Phospholipids with saturated acyl chains show slightly lowered melting temperatures and reduced cooperativity of melting upon CaATPase insertion. In addition, protein induces (at most) slight disorder into the acyl chains at temperatures removed from the lipid melting point. (3) The strongest response is observed for phospholipids containing one saturated and one unsaturated chain (POPE or SOPC) or heterogeneous systems with low levels of unsaturation (egg sphingomyelin). In these cases, relatively low protein levels diminish the magnitude of or completely abolish the phospholipid phase transition. In addition, substantial disorder is introduced into the acyl chain compared with the pure lipid both above and below its transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein transfer between A-I-containing lipoprotein subpopulations: evidence of non-transferable A-I in particles with A-II.

Transfer of apolipoproteins (apo) between the two subpopulations of apo A-I-containing lipoproteins in human plasma: those with A-II [Lp(AI w AII)] and those without [Lp(AI w/o AII)], were studied by observing the transfer of 125I-apo from a radiolabeled subpopulation to an unlabeled subpopulation in vitro. When Lp(AI w AII) was directly radioiodinated, 50.3 +/- 7.4 and 19.5 +/- 7.7% (n = 6) of the total radioactivity was associated with A-I and A-II, respectively. In radioiodinated Lp(AI w/o AII), 71.5 +/- 6.8% (n = 6) of the total radioactivity was A-I-associated. Time-course studies showed that, while some radiolabeled proteins transferred from one population of HDL particles to another within minutes, at least several hours were necessary for transfer to approach equilibrium. Incubation of the subpopulations at equal A-I mass resulted in the transfer of 51.8 +/- 5.0% (n = 4) of total radioactivity from [125I]Lp(AI w/o AII) to Lp(AI w AII) at 37 degrees C in 24 h. The specific activity (S.A.) of A-I in the two subpopulations after incubation was nearly identical. Under similar incubation conditions, only 13.4 +/- 4.6% (n = 4) of total radioactivity was transferred from [125I]Lp(AI w AII) to Lp(AI w/o AII). The S.A. of A-I after incubation was 2-fold higher in particles with A-II than in particles without A-II. These phenomena were also observed with iodinated high-density lipoproteins (HDL) isolated by ultracentrifugation and subsequently subfractionated by immunoaffinity chromatography. However, when Lp(AI w AII) radiolabeled by in vitro exchange with free [125I]A-I was incubated with unlabeled Lp(AI w/o AII), the S.A. of A-I in particles with and without A-II differed by only 18% after incubation. These data are consistent with the following: (1) in both populations of HDL particles, some radiolabeled proteins transferred rapidly (minutes or less), while others transferred slowly (hours); (2) when Lp(AI w AII) and Lp(AI w/o AII) were directly iodinated, all labeled A-I in particles without A-II were transferable, but some labeled AI in particles with A-II were not; (3) when Lp(AI w AII) were labeled by in vitro exchange with [125I]A-I, considerably more labeled A-I were transferable. These observations suggest the presence of non-transferable A-I in Lp(AI w AII).     

Role of apolipoprotein A-I in the structure of human serum high density lipoproteins. Reconstitution studies.

For a better definition of the role of human serum apolipoprotein A-I (apo A-I) in high density lipoprotein structure, a systematic investigation was carried out on factors influencing the in vitro association of this apoprotein with lipids obtained from the parent high density lipoprotein (HDL); these lipids include phospholipids, free cholesterol, cholesteryl esters, and triglycerides. Following equilibration, mixtures of apo A-I and lipids in varying stoichiometric amounts were fractionated by sequential flotation, CsCl density gradient ultracentrifugation, or gel-permeation chromatography, and the isolated complexes were characterized by physicochemical means. As defined by operational criteria (flotation at density 1,063 to 1.21 g/ml), only two types of HDL complexes were reassembled; one, reconstituted HDLS, small with a radius of 31 A, and the other, reconstituted HDLL, large with a radius of 39 A. The two types incorporated all of the lipid constituents of native HDL and contained 2 and 3 mol of apo A-I, respectively. A maximal yield of reconstituted HDL (R-HDL) was observed at an initial protein concentration of 0.1 muM, where apo A-I is predominantly monomeric. At increasing protein concentrations, the amount of apo A-I recovered in R-HDL was found to be proportional to the initial concentration of monomer and dimer in solution. The composition and yield of the complexes were independent of ionic strength and pH within the ranges studied. Both simple incubation and cosonication of apo A-I with HDL phospholipids produced complexes of identical composition, although the yeild of complexes was higher with co-sonication. When the comparison of the same methods was extended to mixtures of apo A-I and whole HDL lipids, the results confirmed previous observations that co-sonication is essential for the incorporation of the neutral lipid into the R-HDL complexes. The results indicate that (a) in vitro complexation of apo A-I with lipids is under kinetic control; (b) apo A-I can generate a lipid-protein complex with properties similar to those of the parent lipoprotein; (c) the process requires well defined experimental conditions and, most importantly, the presence in solution of monomers and dimers of apo A-I; (d) the number of apo A-I molecules incorporated into R-HDL determines the size and structure of the reassembled particle. All of these observations strongly support the essential role of apo A-I in the structure of human HDL.     

Covalent binding of photosensitive 1-(12-azido-[9,10(-3)H2]oleoyl)glycero-3-phosphocholine (lysolecithin) to human serum high density apolipoproteins.

Human serum high density apoproteins were complexed with increasing concentrations of 1-(12-azido-[9,10(-3)H2]oleoyl)glycero-3-phosphocholine up to the saturation concentration (72 mol lysolecithin per mol apo HDL). Ultraviolet irradiation generated the nitrene which led to crosslinking with the two main apolipoproteins AI and AII. Methods are described for the removal of excess, unbound lipid and the column chromatographic separation of the lipopolypeptides AI and AII.     

Conversion of the lipoprotein secreted by cultured eel hepatocytes to high density lipoprotein

• 1.1. The lipoprotein, a VLDL-like lipoprotein, secreted by cultured eel hepatocytes was incubated with whole eel serum, serum HDL, or serum VLDL. No change in the VLDL-like lipoprotein was found.
• 2.2. The secreted lipoprotein was incubated with five kinds of liposomes and a HDL-like particle was formed in the presence of BSA only when l-α-dimyristoyl lecithin liposome was used.
• 3.3. In the presence of 3% BSA, apo AI, proapo AI, apo AII and apo C of the secreted lipoprotein were transferred to the l-α-dimyristoyl lecithin liposome and a HDL-like particle was formed.
• 4.4. The secreted lipoprotein was hydrolyzed by lipoprotein lipase and a HDL-like particle formed after hydrolysis contained no triglyceride and had phospholipid as its main lipid.

     

Physico-chemical aspects of the interaction between DNA and oppositely charged mixed liposomes

The mechanism of complex formation between DNA and oppositely charged dioctadecyldimethylammonium bromide/dioleoyl phosphatidylethanolamine (DODAB/DOPE) and 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)/DOPE mixed liposomes, as well as the physico-chemical properties of DNA-mixed liposome complexes, were examined. Fluorescence microscopy showed that the interaction between DNA and oppositely charged mixed liposomes started at very low liposome concentrations and induced a discrete coil-globule transition in individual DNA molecules. The DNA size distribution was bimodal in a wide range of liposome concentrations. The critical concentration of the cationic lipid needed for the complete compaction of single DNA molecules depended on the composition of the charged mixed DODAB/DOPE and DOTAP/DOPE liposomes. Cryogenic transmission electron microscopy (cryo-TEM) observations of DNA complexes with mixed liposomes revealed that the lamellar packing of lipid molecules was typical for the complexes formed from the cationic lipid-enriched mixtures, while inverted hexagonal arrays were found for the neutral lipid-enriched complexes. The microstructures of the complexes were also examined with the use of the small-angle X-ray scattering (SAXS) technique, which confirmed the results obtained by cryo-TE microscopy and enabled the quantitative characterization of lipid packaging in the complexes with DNA macromolecules. We also found that the introduction of the neutral lipid into the complexes between DNA and oppositely charged lipids, DODAB and DOTAP, moderately increased the thermal stability of the complexes and changed the quantitative characteristics of the melting profiles of the complexes.     

Immunological relationship between bile lipoprotein complex and high density lipoprotein.

Apo bile lipoprotein complex was isolated from human gallbladder bile and an antiserum was prepared. Double immunodiffusion studies show that apo bile lipoprotein complex is present in human plasma and more precisely one of its proteic component is common to bile lipoprotein complex and HDL fraction. This proteic component is different from apo AI, AII, apo B, apo CI, CII, CIII, apo D, apo E and albumin.     

Effect of streptolysin S on liposomes. Influence of membrane lipid composition on toxin action

The effect of the bacterial cytolytic toxin, streptolysin S, on liposomes composed of various phospholipids was investigated. Large unilamellar vesicles containing [14C]sucrose were prepared by reverse-phase evaporation, and membrane damage produced by the toxin was measured by following the release of labeled marker. The net charge of the liposomes had little or no effect on their susceptibility to steptolysin S and the toxin was about equally effective on liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylglycerol. Experiments with liposomes composed of synthetic phospholipids showed that the ability of the toxin to produce membrane damage depended on the degree of unsaturation of the fatty acyl chains. The order of sensitivity was C18 : 2 phosphatidylcholine greater than C18: I phosphatidylcholine greater than C18 : 0 phosphatidylcholine = C16 : 0 phosphatidylcholine. Liposomes containing the latter two phospholipids were virtually unaffected by streptolysin S, and experiments with C18 : 0 phosphatidylcholine suggested that toxin activity does not bind to liposomes composed of phospholipids with saturated fatty acyl chains. The inclusion of 40 mol% cholesterol in C16 : 0 phosphatidylcholine and C18 : 0 phosphatidylcholine liposomes made these vesicles sensitive to streptolysin S. Egg phosphatidylcholine liposomes, which were unaffected at 0 degrees C and 4 degrees C became susceptible to the toxin at these temperatures when cholesterol was included. Liposomes composed of C14 : 0 phosphatidylcholine were unaffected by streptolysin S at temperatures below the chain-melting transition temperature (23 degrees C) of this phospholipid, but became increasingly susceptible above this temperature. The results suggest that the fluidity of the phospholipid hydrocarbon chains in the membrane is important in streptolysin S action.     

Synthetic amphipathic peptides resembling apolipoproteins stimulate the release of human placental lactogen

Previous studies from our laboratory demonstrated native high density lipoproteins and apolipoproteins AI, AII, and CI, stimulate the release of human placental lactogen (hPL) from trophoblast cells in culture. To examine the mechanisms by which these apolipoproteins stimulate hPL release, we have studied hPL secretion in response to several synthetic peptide analogs of the amphipathic helical structure of the apolipoproteins. The magnitude of the stimulation of hPL release in response to the analog peptides correlated with the ability to displace apolipoproteins from high density lipoprotein and with other measures of phospholipid binding affinity such as the increase in alpha-helicity and the size of complexes formed between the peptide and phospholipid. The correlation of stimulatory ability and lipid affinity suggests that the action of the apolipoproteins on hPL release may be mediated through an interaction with plasma membrane phospholipids.     

Physical studies on membrane lipids of Bacillus stearothermophilus temperature and calcium effects

Bacillus stearothermophilus was grown at the optimal temperature range (center, 65 degrees C), below it (48 and 55 degrees C), and above it (68 degrees C), in a complex medium with or without 2.5 mM Ca2+. The Ca(2+)-supplement improves growth at sub- and supraoptimal temperatures and extends it to higher temperatures (Jurado et al. (1987) J. Gen. Microbiol. 133, 507-513). The phospholipid composition of cultures obtained in the different growth conditions was studied. Phosphatidylethanolamine was always the major phospholipid (40 to 50% of the total phospholipid). Diphosphatidylglycerol, phosphatidylglycerol, a phosphoglycolipid (pgl) and two minor phospholipids (not identified) were also found in the polar lipid extract. The pgl shows a threefold concentration increase as the growth temperature raises from 48 to 68 degrees C. The thermotropic behavior of membrane lipids was studied by differential scanning calorimetry (DSC) and by means of two fluorescent probes of fluidity, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1,3-di(2-pyrenyl)propane (2Py(3)2Py). The results reveal similar features and clearly show a shift of the temperature range of the phase transition to higher values and an increased structural order of the bilayer, as the growth temperature rises from 55 to 68 degrees C, but an opposite effect was observed from 48 to 55 degrees C. Although the Ca(2+)-supplement to the growth medium has no detectable effect, the addition of Ca2+ to the buffer of liposomes (Ca(2+)-liposomes) has a significant ordering effect at all growth temperatures. These liposomes show a shift of the transition range to higher temperatures and the fluorescent parameters (DPH polarization and intramolecular excimerization of the 2Py(3)2Py) detected an order increase of the probes environment, along and above the main phase transition. Spectra of 31P-NMR and polarized light microscopy clearly show that the lipid extracts exhibit, in all the conditions, typical lamellar phase geometry. We concluded that B. stearothermophilus controls the membrane lipid composition to compensate for the destabilizing effect of high temperatures on the membrane organization or to provide an appropriate packing of phospholipid molecules in a stable bilayer. At high temperatures, Ca(2+)-stimulatory effect on growth is presumably due to a direct Ca2+ interaction with the membrane phospholipids, inducing an increased structural order on the bilayer. The increase of the phase transition temperature in the total lipid extracts as compared with the respective polar lipid fractions probably indicates a stabilizing effect of neutral lipids on membrane bilayers.     

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid.(ABSTRACT TRUNCATED AT 400 WORDS)