Regulation of transbilayer distribution of a fluorescent sterol in tumor cell plasma membranes

The lipid composition and transbilayer distribution of plasma membrane isolated from primary tumor (L-929, LM, A-9 and C3H) and nine metastatic cell lines cultured under identical conditions was examined. Cultured primary tumor and metastatic cells differed two-fold in sterol/phospholipid molar ratios. There was a direct correlation between plasma membrane anionic phospholipid (phosphatidylinositol and phosphatidylserine) content and plasma membrane sterol/phospholipid ratio. This finding may bear on the possible link between oncogenes and inositol lipids. The fluorescent sterol, dehydroergosterol, was incorporated into primary tumor and metastatic cell lines. Selective quenching of outer monolayer fluorescence by covalently linked trinitrophenyl groups demonstrated an asymmetric transbilayer distribution of sterol in the plasma membranes. The inner monolayer of the plasma membranes from both cultured primary and metastatic tumor cells was enriched in sterol as compared with the outer monolayer. Consistent with this, the inner monolayer was distinctly more rigid as determined by the limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Dehydroergosterol fluorescence was temperature dependent and sensitive to lateral phase separations in phosphatidylcholine vesicles and in LM cell plasma membranes. Dehydroergosterol detected phase separations near 24 degrees C in the outer monolayer and at 21 degrees C and 37 degrees C in the inner monolayer of LM plasma membranes. Yet, no change in transbilayer sterol distribution was detected in ascending or descending temperature scans between 4 and 45 degrees C. Alterations in plasma membrane phospholipid polar head group composition by choline analogues (N,N-dimethylethanolamine, N-methylethanolamine, and ethanolamine) also did not perturb transbilayer sterol asymmetry. Treatment with phenobarbital or prilocaine, drugs that selectively fluidize the outer and inner monolayer of LM plasma membranes, respectively, did not change dehydroergosterol transbilayer distribution.     

Sorting of lipids and transmembrane peptides between detergent-soluble bilayers and detergent-resistant rafts

Specific proteins and lipids sequester to regions of cell membranes called rafts. Due to their high content of sphingomyelin (SM) and cholesterol, raft bilayers are thicker than nonraft bilayers and, at least at 4 degrees C, are resistant to Triton X-100 extraction. It has been postulated that rafts concentrate proteins with long transbilayer domains because of "hydrophobic matching" between the transbilayer domain and the thick bilayer hydrocarbon region. However, because the area compressibility and bending moduli of SM:cholesterol bilayers are larger than that of nonraft bilayers, there should be an energy cost to partition proteins or peptides into rafts. To determine the effects on peptide sorting of raft thickness and mechanical properties, we incorporated two transbilayer peptides (P-23, P-29) into bilayers composed of SM, dioleoylphosphatidylcholine, and cholesterol, separated detergent-soluble membranes (DSMs) from detergent-resistant membranes (DRMs), and measured their peptide and lipid compositions. P-23 and P-29 were designed to have transbilayer domains that matched the hydrocarbon thicknesses of DSMs and DRMs, respectively. At both 4 degrees C and 37 degrees C DSMs were enriched in dioleoylphosphatidylcholine and DRMs were enriched in SM and cholesterol. At both temperatures both P-23 and P-29 preferentially localized to DSMs, demonstrating the importance of bilayer mechanical properties relative to hydrophobic mismatch. However, at 37 degrees C significantly more P-29 than P-23 was located in DRMs, implying that hydrophobic matching played a role in peptide sorting at physiological temperature. These experiments demonstrate that the sorting of peptides as measured by detergent extraction is temperature-dependent and both bilayer mechanical properties and hydrophobic matching impact peptide distribution between DSMs and DRMs.     

Plasmid pR386 renders Escherichia coli cells restrictive to the growth of bacteriophage T4 unf mutants.

The introduction of the F1 incompatibility group plasmid pR386 Tc into several common laboratory strains of Escherichia coli rendered them restrictive to the growth of bacteriophage T4 unf mutants, which are defective in unfolding the host genome. The growth inhibition was temperature dependent. The single mutant unf39 x 5 exhibited an efficiency of plating of less than 10(-8) at 27 degrees C. However, at 37 degrees C, complete growth inhibition occurred only when host DNA degradation was also absent.     

Dipyrenylphosphatidylcholines as membrane fluidity probes. Relationship between intramolecular and intermolecular excimer formation rates.

In the intramolecular excimeric membrane probe, dipyrenylphosphatidylcholine (dipyn PC), pyrene moieties are linked to the terminal carbons of the two acyl chains, each of which contains n carbons. We show here how the probe intramolecular excimer production rate, K, may be determined from the excimer/monomer intensity ratio, rl, by making use of the fluorescence titrations of the related monopyrenyl probe, pyn PC, analyzed according to the milling crowd model. rl and the rate K of dipy10 PC in four model membrane systems were measured over a wide temperature range and both parameters are shown to be sensitive functions of the lateral fluidity of the host matrix. A model for relating the intramolecular and intermolecular excimer formation rates is proposed according to which both processes are limited by the reorientational rate of the pyrene moiety. Above the fluid-gel transition temperature, Tc, the diffusion rate (f) of the monopyrenyl probe (pyn PC) is accordingly related to K by: pE approximately K/(K + 1/2f + tau -1M), where pE is the probability of excimer formation between nearest neighbor pyn PC probes, and tau M is the monomer lifetime. Values of pE derived in this way are found to be consistent with pE values derived from the milling crowd analysis of fluorescence yield titration experiments. K for dipy10 PC in DMPC multibilayers ranges from 0.21 x 10(7) s-1 at 10 degrees C in the gel phase, to 5.7 x 10(7) s-1 at 60 degrees C in the fluid phase, whereas the lateral diffusion coefficient, D, for py10 PC in the same bilayers ranged from 8 to 34 microns2 s-1, when calculated with D = fL2/4, L being the average lipid-lipid spacing of the host membrane. Above Tc and at the same reduced temperature, (T - Tc)/Tc, both f for py10 PC, and K for dipy10 PC were found to have relative magnitudes in the order: DPPC greater than DMPC greater than POPC greater than DOPC. This and the similarity of the activation energies for f and K suggest that the rotation of the the pyrene moiety is the rate-limiting step for both the lateral mobility of py10 PC and intramolecular excimer formation in dipy10 PC.     

Lateral diffusivity of lipid analogue excimeric probes in dimyristoylphosphatidylcholine bilayers.

The lateral mobility of pyrenyl phospholipid probes in dimyristoylphosphatidylcholine (DMPC) vesicles was determined from the dependence of the pyrene monomeric and excimeric fluorescence yields on the molar probe ratio. The analysis of the experimental data makes use of the milling crowd model for two-dimensional diffusivity and the computer simulated random walks of probes in an array of lipids. The fluorescence yields for 1-palmitoyl-2-(1'-pyrenedecanoyl)phosphatidylcholine (py10PC) in DMPC bilayers are well fitted by the model both below and above the fluid-gel phase transition temperature (Tc) and permit the evaluation of the probe diffusion rate (f), which is the frequency with which probes take random steps of length L, the host membrane lipid-lipid spacing. The lateral diffusion coefficient is then obtained from the relationship D = fL2/4. In passing through the fluid-gel phase transition of DMPC (Tc = 24 degrees C), the lateral mobility of py10PC determined in this way decrease only moderately, while D measured by fluorescence photobleaching recovery (FPR) experiments is lowered by two or more orders of magnitude in gel phase. This difference in gel phase diffusivities is discussed and considered to be related either to (a) the diffusion length in FPR experiments being about a micrometer or over 100 times greater than that of excimeric probes (approximately 1 nm), or (b) to nonrandomicity in the distribution of the pyrenyl probes in gel phase DMPC. At 35 degrees C, in fluid DMPC vesicles, the diffusion rate is f = 1.8 x 10(8) s-1, corresponding to D = 29 microns2 s-1, which is about three times larger than the value obtained in FPR experiments. The activation energy for lateral diffusion in fluid DMPC was determined to be 8.0 kcal/mol.     

Properties of spin labelled membranes of Fusarium oxysporum f. sp. lycopersici.

Growth temperature-induced compositional changes in membranes of Fusarium oxysporum provided a test system for study of the relationship between physical properties and composition. Growth at 15 degrees C was characterized by a decrease in phospholipid content relative to sterol content, a shift in phospholipid composition from phosphatidylcholine to phosphatidylethanolamine and a marked enhancement in the amount of polyunsaturated fatty acids in the phospholipid and triglyceride classes. Uptake of a spin labelled analog of stearic acid during growth and subsequent solution of the probe in the membranes allowed estimation of viscosity and molecular order of the membranes of live cells and of isolated membrane preparations. Less than 1/20 of the intracellular label was accessible to sodium ascorbate while none was released by sodium dodecyl sulfate. All of the label in live cells was reduced by in vivo respiratory activity above 20 degrees C but this process could be reversed or avoided by added ferricyanide. A cholestane spin probe was also incorporated into the membranes. The probes were not reduced as readily in isolated membranes and hence fluidity of the membranes could be assessed over a wide temperature range. At low temperatures (-10 degrees C) a nonlethal, liquid-solid phase transition was indicated in isolated membrane lipids while at higher (lethal) temperatures (40-45 degrees C), discontinuities appeared in Arrhenius plots of rotational correlation time. Activation energies for isotropic rotation of the stearate probes in the membranes changed markedly in this temperature range and this effect correlated closely with loss of viability of conidial cells. Correlation times for stearate probes showed little variation with growth temperature nor were any breaks in Arrhenius plots of this parameter detected in the range 0-35 degrees C in whole cells or isolated membranes. The data indicated control of membrane physical properties within close tolerances throughout the physiological temperature range regardless of growth temperature. It was concluded that this homeostatic phenomenon was due to the counteractive effects of sterol/phospholipid ratio, phospholipid composition and fatty acid polyunsaturation since the condensing and fluidizing components of the isolated total membranes vary in a reciprocal manner.     

Rapid transbilayer movement of spin-labeled steroids in human erythrocytes and in liposomes

The transbilayer movement and distribution of spin-labeled analogs of the steroids androstane (SLA) and cholestane (SLC) were investigated in the human erythrocyte and in liposomes. Membranes were labeled with SLA or SLC, and the analogs in the outer leaflet were selectively reduced at 4C using 6-O-phenylascorbic acid. As shown previously, 6-O-phenylascorbic acid reduces rapidly nitroxides exposed on the outer leaflet, but its permeation of membranes is comparatively slow and thus does not interfere with the assay. From the reduction kinetics, we infer that transbilayer movement of SLA in erythrocytes is rapid at 4C with a half-time of approximately 4.3 min and that the probe distributes almost symmetrically between both halves of the plasma membrane. We have no indication that a protein-mediated transport is involved in the rapid transbilayer movement of SLA because 1) pretreatment of erythrocytes with N-ethyl maleimide affected neither flip-flop nor transbilayer distribution of SLA and 2) flip-flop of SLA was also rapid in pure lipid membranes. The transbilayer dynamics of SLC in erythrocyte membranes could not be resolved by our assay. Thus, the rate of SLC flip-flop must be on the order of, or even faster than, that of probe reduction rate on the exoplasmic leaflet (half-time approximately 0.5 min). The results are discussed with regard to the transbilayer dynamics of cholesterol.     

Involvement of the protein-protein interactions in the thermodynamics of the electron-transfer process in the reaction centers from Rhodopseudomonas viridis

Reaction centers from Rhodopseudomonas viridis were reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) liposomes. Freeze-fracture electron micrographs were performed on the samples frozen from temperatures above and below the phase transition temperatures of those lipids (Tc = 23 and 9.5 degrees C, in DMPC and DEPC, respectively). Above Tc, in the fluid conformation of the lipids, the reaction centers are randomly distributed in the vesicle membranes. Below Tc, aggregation of the proteins occurs. The Arrhenius plots of the rate constants of the charge recombination between P+ and QA- display a break at about 24 degrees C in DMPC vesicles and about 10 degrees C in DEPC vesicles (P represents the primary electron donor, a dimer of bacteriochlorophyll, and QA the primary quinone electron acceptor). This is in contrast to what was previously observed for the proteoliposomes of egg yolk phosphatidylcholine and for chromatophores [Baciou, L., Rivas, E., & Sebban, P. (1990) Biochemistry 29, 2966-2976], for which Arrhenius plots were linear. In DMPC and DEPC proteoliposomes, the activation parameters were very different on the two sides of Tc (delta H degrees for T less than Tc = 2.5 times delta H degrees for T greater than Tc), leading however, to the same delta G degrees values. Taking into account the structural and thermodynamic data, we suggest that, in vivo, protein-protein interactions play a role in the thermodynamic parameters associated with the energy stabilization process within the reaction centers.     

The effect of acclimation temperature on the fusion kinetics of lipid vesicles derived from endoplasmic reticulum membranes of rainbow trout (Oncorhynchus mykiss) liver

Membrane fusion is an obligatory step in many vital cellular processes. The well-established enrichment of bilayer-destabilizing lipids in membranes of poikilotherms subjected to growth at low temperatures leads to the prediction that such membranes will possess a greater propensity to undergo fusion. This hypothesis was explicitly tested in the present study by determining the kinetics of fusion between small unilamellar vesicles (SUVs) prepared from endoplasmic reticulum (ER) membranes of thermally-acclimated (to 5 and 20 degrees C) rainbow trout (Oncorhynchus mykiss) liver and bovine brain phosphatidylserine (BBPS). At temperatures above 10 degrees C, ER vesicles from 5 degrees C-acclimated trout, fused more rapidly and to a greater extent with BBPS vesicles (by average factors of 1.25- and 1.45-fold, respectively) than ER vesicles of 20 degrees C-acclimated trout. At temperatures >35 degrees C, apparent fusion rates declined while the extent of fusion increased in both acclimation groups. Fusion kinetics were found to be well correlated with and limited by the physical properties and phase state of the BBPS vesicles. These results indicate that dynamic attributes of biological membranes, such as the propensity to undergo fusion, are of potential regulatory significance and are partially conserved when growth or environmental temperature changes.     

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

Fluorescence measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in native Tetrahymena pyriformis microsomal membranes revealed characteristic "break points" in curves of polarization vs. temperature. In the 5--35 degree C range, membranes from cells grown at 39 degrees C exhibited two break points, one at 11.6 +/- 0.6 degrees C and another at 23.1 +/- 1.6 degrees C. Membranes from 15 degrees C grown cells also showed two break points, one at 8.0 +/- 1.7 degrees C and another at 17.7 +/- 1.7 degrees C. Complementary measurements of turbidity (absorbance at 360 nm) vs. temperature revealed break points at approximately the same temperatures as observed with the fluorescent probe, thus strengthening the likelihood that the break points signify the onset or termination of lipid phase separations or some other significant structural alteration of lipids. In general, break points measured in the native membrane samples occurred at slightly lower temperatures than did break points in lipids extracted from comparable membranes. This suggests two possible types of protein--lipid interaction. First, there may be a selective withdrawal of relatively highly saturated phospholipid molecular species from the bulk lipid phase and into protein annulus regions. Alternatively, the configuration of the hydrophobic core of certain key membrane proteins may be such that nonspecific interactions with the lipids stabilize the liquid-crystalline phase.     

Unusual pressure dependence of the lateral motion of pyrene-labeled phosphatidylcholine in bipolar lipid vesicles.

The lateral mobility of a pyrene-labeled phosphatidylcholine probe in liposomes containing archaebacterial bipolar lipids has been studied isothermally as a function of pressure. The pressure-dependence of the probe mobility, R, is found to be slightly positive or zero in the temperature range of 17 - 48 degrees C. At temperatures > 48 degrees C, R becomes negative and decreases with temperature. The data indicate that lateral mobility only becomes appreciable at high temperatures. In addition, the R values obtained with other lipid membranes are much lower than that obtained with bipolar liposomes, implying that the membranes of archaebacterial liposomes are laterally immobile, as compared to other lipid membranes.     

Temperature dependence of membrane ion conductance analyzed by using the amphiphilic anion 5/6-carboxyfluorescein

The rate of efflux of trapped 5/6-carboxyfluorescein from sealed lipid vesicles showed a marked dependence on (a) temperature, (b) phospholipid acyl chain composition, and (c) the nature of co-trapped counterions. When the dye was salted with sodium, at pH greater than 7, the rate of dye permeation showed a discrete maximum at the melting point of the lipid bilayer (Tc); in the case of membranes composed of dipalmitoylphosphatidylcholine, this discontinuity extended over a very broad temperature range, being detectable at least 10 degrees C above and below Tc. The peak in dye permeation rate was superimposed on a permeation profile that showed a simple exponential relationship to temperature. Studies with a homologous series of saturated lecithin bilayers revealed a consistent pattern of behavior: a logarithmic dependence of dye permeation rate on temperature with a superimposed discontinuity at Tc. For thin membranes (12-14-carbon acyl chains), the discontinuity was severe, exerting an influence over a very broad temperature range and leading to extremely high overall dye leakage rates. As the acyl chains were lengthened, the discontinuity became less pronounced, almost disappearing at a chain length of 20 carbons. In sharp contrast to these results, dye salted with N-methylglucamine [or with tris(hydroxymethyl)aminomethane] showed no efflux maximum at Tc, and base-line leakage rates were generally slower. When dye was salted with ammonium, efflux was too rapid to monitor, even at temperatures well below Tc. The results indicate that the rate of release of electrically charged dyes, such as 5/6-carboxyfluorescein, from sealed lipid vesicles can be tightly coupled to the counterion leakage rate and hence can provide an accurate and convenient assay of relative ion flux across phospholipid bilayers.     

Cross-linking studies of the self-association properties of apo-A-I and apo-A-II from human high density lipoprotein.

The state of self-association of the apoprotein components of human high density lipoprotein have been studied by use of the cross-linking reagent dimethyl-suberimidate. Analusis of the cross-linked products was carried out by soduim dodecyl sulfate-gel electrophoresis and by agarose column chromatography in 6 M guanidine hydrochloride. Apo-A-I was found to exist as a monomer at low concentration, but associates to tetrameric and pentameric forms at concentrations of 0.5 mg/ml or higher. The self-association was found to be ionic strength-dependent, with association promoted by the presence of salt. Apo-A-II was also found to associate, but the major oligomeric form observed was dimeric (Mr = 34,000), and the association was less dependent on ionic strength than for apo-A-I. Cross-linking in the presence of various concentrations of guanidine hydrochloride showed that apo-A-II self-association persisted at higher concentrations of the denaturant than for apo-A-I. Studies of the effect of temperature demonstrated that the self-association of both proteins was diminished at temperatures above 30 degrees C. Recombination of apo-A-II with phospholipid resulted in the formation of particles which yielded primarily trimers upon cross-linking. This suggests that phospholipid binding causes major reorganization of the self-associated forms of apo-A-II.     

Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach.

The effects of cholesterol on the structure and dynamics of dimyristoylphosphatidylcholine (DMPC) model membranes have been monitored as functions of temperature and cholesterol concentration in the membrane. The use of deuterium labels both on the cholesterol fused ring system and on the lipid chains in conjunction with solid state deuterium nuclear magnetic resonance (2H-NMR) afforded to monitor the degree of ordering of both molecules in a mixed system. The degree of ordering of the lipid head group was followed by phosphorus-31 (31P)-NMR. New findings on the effect of cholesterol on DMPC may be summarized as follows: i) cholesterol disorders the lipid chains below temperature of the DMPC gel-to-fluid transition (Tc) and orders them above; the effect is linear with cholesterol concentration at 0 and 60 degrees C but for intermediate temperatures, a saturation effect is observed at 20-30 mol %; ii) the ordering-disordering effects are perceived similarly by all chain segments with, however, a greater sensitivity for positions near the bilayer center; iii) below Tc, the lipid head group is considerably disordered by increasing amounts of cholesterol but slightly affected above; iv) the degree of ordering of cholesterol is quasi temperature independent for fractions greater than or equal to 30%; v) the average orientation of the cholesterol rigid body is perpendicular to the bilayer surface and exhibits little variations with temperature and cholesterol concentration. Variations in membrane dynamics are interpreted in terms of cholesterol-induced changes in bilayer thickness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Composition and physical properties of lipids from plasma membranes of dog kidney

Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have been determined in plasma membranes of the dog kidney. The lipid composition of brush-border membranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospholipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44 degrees C approximately. In the range of temperature studied (5-55 degrees C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37 degrees C which compares with that of other biological membranes.     

Morphological behavior of lipid bilayers induced by melittin near the phase transition temperature

Morphological changes of DMPC, DLPC, and DPPC bilayers containing melittin (lecithin/melittin molar ratio of 10:1) around the gel-to-liquid crystalline phase transition temperatures (Tc) were examined by a variety of biophysical methods. First, giant vesicles with the diameters of approximately 20 microm were observed by optical microscopy for melittin-DMPC bilayers at 27.9 degrees C. When the temperature was lowered to 24.9 degrees C (Tc = 23 degrees C for the neat DMPC bilayers), the surface of vesicles became blurred and dynamic pore formation was visible in the microscopic picture taken at different exposure times. Phase separation and association of melittin molecules in the bilayers were further detected by fluorescent microscopy and mass spectrometry, respectively. These vesicles disappeared completely at 22.9 degrees C. It was thus found that the melittin-lecithin bilayers reversibly undergo their fusion and disruption near the respective Tcs. The fluctuation of lipids is, therefore, responsible for the membrane fusion above the Tc, and the association of melittin molecules causes membrane fragmentation below the Tc. Subsequent magnetic alignments were observed by solid-state (31)P NMR spectra for the melittin-lecithin vesicles at a temperature above the respective Tcs. On the other hand, additional large amplitude motion induced by melittin at a temperature near the Tc breaks down the magnetic alignment.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A(2). We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.     

Transbilayer distribution of phospholipids in bacteriophage membranes

We have previously demonstrated that the membranes of several bacteriophages contain more phosphatidylglycerol (PG) and less phosphatidylethanolamine (PE) than the host membrane from where they are derived. Here, we determined the transbilayer distribution of PG and PE in the membranes of bacteriophages PM2, PRD1, Bam35 and phi6 using selective modification of PG and PE in the outer membrane leaflet with sodium periodate or trinitrobenzene sulfonic acid, respectively. In phi6, the transbilayer distributions of PG, PE and cardiolipin could also be analyzed by selective hydrolysis of the lipids in the outer leaflet by phospholipase A₂. We used electrospray ionization mass-spectrometry to determine the transbilayer distribution of phospholipid classes and individual molecular species. In each bacteriophage, PG was enriched in the outer membrane leaflet and PE in the inner one (except for Bam35). Only modest differences in the transbilayer distribution between different molecular species were observed. The effective shape and charge of the phospholipid molecules and lipid-protein interactions are likely to be most important factors driving the asymmetric distribution of phospholipids in the phage membranes. The results of this first systematic study on the phospholipid distribution in bacteriophage membranes will be very helpful when interpreting the accumulating high-resolution data on these organisms.     

Pulmonary surfactant secretion is regulated by the physical state of extracellular phosphatidylcholine.

Pulmonary alveolar type II cells synthesize, secrete, and recycle the components of pulmonary surfactant. In this report we present evidence that dipalmitoylphosphatidylcholine is a potent inhibitor of surfactant lipid secretion by type II cells. Monoenoic and dienoic phosphatidylcholines with fatty acids of 16 or 18 carbons are ineffective as inhibitors of surfactant lipid secretion. In contrast, disaturated phosphatidylcholines, with either symmetric or asymmetric pairs of fatty acids of 14, 16, or 18 carbons, exhibit inhibition of surfactant secretion that correlates extremely well with the phase transition temperature (Tc) of the phospholipid. The inhibitory activity of dipalmitoylphosphatidylcholine is not dependent upon lipid stereochemistry. N-Methylated derivatives of dipalmitoylphosphatidylethanolamine are significantly less effective than phosphatidylcholine as inhibitors. Phosphatidylcholines below their phase transition temperature are inhibitors of surfactant secretion, whereas those above their phase transition temperature are either ineffective or weakly inhibitory. The phase transition dependence of inhibition is observed when type II cells are incubated at 37 degrees C with different species of phosphatidylcholine. In addition, if type II cells are stimulated to secrete at different temperatures the efficacy of a given phospholipid as an inhibitor is dependent on its relationship to Tc (i.e. dipalmitoylphosphatidylcholine with a Tc of 41 degrees C significantly inhibits secretion at 37 degrees C but not at 42 degrees C). Inhibition of surfactant secretion by dipalmitoylphosphatidylcholine is abrogated when it is incorporated into the same liposome with dioleoylphosphatidylcholine as a 50:50 mixture. In contrast, the simultaneous addition of two separate populations of liposomes, one composed of dipalmitoylphosphatidylcholine and the other composed of dioleoylphosphatidylcholine, does not significantly alter the inhibitory activity found with dipalmitoylphosphatidylcholine alone. These data provide compelling evidence that the physical state of phosphatidylcholine can regulate surfactant secretion from alveolar type II cells and suggest a unique mechanism for regulating exocytosis in the alveolus of the lung.