Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15 degrees C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37 degrees C membranes, while 15 degrees C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15 degrees C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15 degrees C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37 degrees C, but only 50% at 15 degrees C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15 degrees C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Bilayer stabilization of phosphatidylethanolamine by N-biotinylphosphatidylethanolamine.

We have examined the ability of biotinylated phosphatidylethanolamine and similar lipids to stabilize the bilayer phase of polymorphic dioleoylphosphatidylethanolamine (DOPE). Sonicated lipid mixtures were characterized in terms of their aggregation state, size and ability to encapsulate and retain the fluorescent dye, calcein. Titration of DOPE with N-biotinyl-PE indicated that stable liposomes could be produced by sonication of DOPE based dispersions containing N-biotinyl-PE at concentrations greater than 8 mol%. These liposomes were relatively small, could efficiently encapsulate calcein, and showed minimal leakage upon prolonged storage at 4 degrees C. Maleimido-4-(p-phenylbutyrate)-PE (MPB-PE) was equally effective at stabilizing the bilayer phase of DOPE whereas N-dinitrophenyl-PE and N-(dinitrophenyl-caproyl)-PE were relatively poor stabilizers, requiring at least 15 mol% for stabilization at pH 7.4. Differential scanning calorimetry of dielaidoylphosphatidylethanolamine (DEPE)/N-biotinyl-PE mixtures indicated that stabilizer concentrations as low as 2 mol% could abolish the L alpha/HII phase transition of DEPE.     

A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium

The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in S?o Pedro do Sul, Portugal, were fractionated by chromatography on silica gel. The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction. The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA). Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction. All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C. Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined. These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL. The significance of these results to the thermophily of the bacterium is discussed.     

Effects of temperature and monovalent cations on activity and quaternary structure of tryptophanase

Effects of temperature and monovalent cations on the activity and the quaternary structure of tryptophanase of Escherichia coli were studied. The conversion of the apoenzyme into the active holoenzyme was attained at 30 degrees C in Tris-HCl buffer (pH 8.0) containing pyridoxal-P and K+, while no conversion occurred at 5 degrees C. The active holoenzyme thus formed was stable even at 5 degrees C, as long as the cation was present. When K+ was absent, however, the active enzyme gradually lost the activity upon chilling to 5 degrees C. The HPLC gel filtration analysis of the active holoenzyme and the low temperature-inactivated enzyme species revealed that the tetrameric holoenzyme dissociated into the dimeric apoenzyme concomitant with the low temperature-induced inactivation at 5 degrees C. The results of HPLC experiments together with other available evidence also suggest that the inactive tetrameric holoenzyme was first formed from the dimeric apoenzyme and pyridoxal-P prior to the formation of the active holoenzyme and that the cation promoted the conversion of the inactive holoenzyme into the active holoenzyme rather than being involved in the conversion of the apoenzyme and pyridoxal-P into the holoenzyme. Among various cations tested for the above effects, NH4+ exhibited the largest effect and K+ the second.     

Interaction of cytochrome P-450 with phospholipids in dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol mixtures

ESR and microcalorimetry methods were employed to investigate the thermotropic properties and structure of proteoliposomes that incorporate cytochrome P450 and DMPC-DMPG binary mixtures depending on cytochrome P450 content and phospholipid composition. The microcalorimetry data demonstrated that the incorporation of cytochrome P450 into the phospholipid mixture resulted in bilayer thermal stabilization. The maximum shift of the temperature and proteoliposome transition enthalpy were achieved at the protein/lipid molar ratio of 1:1000 in almost equimolar phospholipid mixture. Using fatty acids that were spin-labeled at different positions (C5, C12, C16), it has been shown that the incorporation of cytochrome P450 into lipid mixtures containing 0-100% DMPG decreases C12 and C16 mobility and increases the C5 order parameter at transition phase (30 degrees C) and liquid crystal phase (37 degrees C) of bilayer. The maximum alteration amplitude of the probes used was not characteristic for the separate DMPC and DMPG but rather for the mixture at the molar ratio close to equimolar value. It is proposed that cytochrome P450 incorporation into the binary mixture initiated the formation of the bilayer crystal-like phase.     

Influence of vitamin E on phosphatidylethanolamine lipid polymorphism

The effect of vitamin E, in its major form alpha-tocopherol and its synthetic analog alpha-tocopheryl acetate, on phosphatidylethanolamine lipid polymorphism has been studied by mean of differential scanning calorimetry and 31P-nuclear magnetic resonance techniques. From the interaction of these tocopherols with dielaidoylphosphatidylethanolamine it is concluded that both molecules promote the formation of the hexagonal HII phase at temperatures lower than those of the pure phospholipid. When the tocopherols were incorporated in the saturated dimiristoylphosphatidylethanolamine, which has been shown not to undergo bilayer to hexagonal HII phase transition, up to 90 degrees C, they induce the phospholipid to partially organize in hexagonal HII phase. From our experiments it is shown that alpha-tocopherol is more effective than its analog in promoting HII phase in these systems. It is also shown that, while alpha-tocopheryl acetate does not significantly perturb the gel to liquid-crystalline phase transition of dimirystoylphosphatidylethanolamine, alpha-tocopherol does so and more than one peak appears in the calorimetric profile, indicating that lateral phase separations are taking place.     

Different effects of long- and short-chain ceramides on the gel-fluid and lamellar-hexagonal transitions of phospholipids: a calorimetric, NMR, and x-ray diffraction study

The effects on dielaidoylphosphatidylethanolamine (DEPE) bilayers of ceramides containing different N-acyl chains have been studied by differential scanning calorimetry small angle x-ray diffraction and (31)P-NMR spectroscopy. N-palmitoyl (Cer16), N-hexanoyl (Cer6), and N-acetyl (Cer2) sphingosines have been used. Both the gel-fluid and the lamellar-inverted hexagonal transitions of DEPE have been examined in the presence of the various ceramides in the 0-25 mol % concentration range. Pure hydrated ceramides exhibit cooperative endothermic order-disorder transitions at 93 degrees C (Cer16), 60 degrees C (Cer6), and 54 degrees C (Cer2). In DEPE bilayers, Cer16 does not mix with the phospholipid in the gel phase, giving rise to high-melting ceramide-rich domains. Cer16 favors the lamellar-hexagonal transition of DEPE, decreasing the transition temperature. Cer2, on the other hand, is soluble in the gel phase of DEPE, decreasing the gel-fluid and increasing the lamellar-hexagonal transition temperatures, thus effectively stabilizing the lamellar fluid phase. In addition, Cer2 was peculiar in that no equilibrium could be reached for the Cer2-DEPE mixture above 60 degrees C, the lamellar-hexagonal transition shifting with time to temperatures beyond the instrumental range. The properties of Cer6 are intermediate between those of the other two, this ceramide decreasing both the gel-fluid and lamellar-hexagonal transition temperatures. Temperature-composition diagrams have been constructed for the mixtures of DEPE with each of the three ceramides. The different behavior of the long- and short-chain ceramides can be rationalized in terms of their different molecular geometries, Cer16 favoring negative curvature in the monolayers, thus inverted phases, and the opposite being true of the micelle-forming Cer2. These differences may be at the origin of the different physiological effects that are sometimes observed for the long- and short-chain ceramides.     

Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase

The structure and thermotropic phase behaviour of aqueous dispersions of dipalmitoylphosphatidylcholine and glucosylceramide rich in C-24 fatty acyl residues was investigated by synchrotron X-ray diffraction methods. Binary mixtures comprised of molar ratios 2.5:100, 6.5:100, 12.6:100, 25:100, 40:100 and 50:100, glucolipid:phospholipid were examined in heating and cooling scans of 2°/min between 25 and 85 °C. Small-angle reflections indicated coexisting lamellar structures over the entire temperature range investigated. Reversible thermotropic changes were observed in one lamellar structure that is consistent with transitions between gel, ripple and fluid lamellar phases of pure phospholipid. The temperature of these transitions, however, were progressively shifted up by about 5 °C in the mixture containing the highest proportion of glucolipid and coincided with a published endothermic peak observed in this mixture. A higher-temperature endotherm was associated with molecular rearrangements on transition of the gel phase phospholipid to the fluid phase. This rearrangement was associated with the appearance of identifiable transient intermediate structures in the small-angle scattering region. The glucolipid formed stoichiometric mixtures with the phospholipid at all temperatures investigated and there was no evidence of phase separation of pure glucolipid. Analysis of the wide-angle scattering profiles during an initial heating scan of a binary mixture comprised of 40:60 glucolipid:phospholipid was consistent with a phase transition of pure phospholipid at about 43 °C coexisting with a liquid-ordered phase formed from the two lipids. This was confirmed by analysis of the small-angle scattering peaks of this mixture recorded at 25 and 65 °C which showed that a glucolipid-rich phase coexisted with almost pure bilayers of phospholipid at both temperatures. The glucolipid-rich phase consisted of 45:55 mole ratio glucolipid:phospholipid at 25 °C with pure phospholipid in gel phase and 42:58 mole ratio at 65 °C when the phospholipid was in the fluid phase. The results are discussed with reference to the role of the length of the N-acyl substituent of the sphingolipids in formation of complexes with phospholipids.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Characterization of a quasicrystalline phase in codispersions of phosphatidylethanolamine and glucocerebroside

Synchrotron x-ray diffraction, differential scanning calorimetry, and electron spin resonance spectroscopy have been employed to characterize a quasicrystalline phase formed in aqueous dispersions of binary mixtures of glucocerebroside and palmitoyloleoylphosphatidylethanolamine. Small- and wide-angle x-ray scattering intensity patterns were recorded during temperature scans between 20 degrees and 90 degrees C from mixtures of composition 2, 5, 10, 20, 30, and 40 mol glucocerebroside per 100 mol phospholipid. The quasicrystalline phase was characterized by a broad lamellar d-spacing of 6.06 nm at 40 degrees C and a broad wide-angle x-ray scattering band centered at approximately 0.438 nm, close to the gel phase centered at approximately 0.425 nm and distinct from a broad peak centered at 0.457 nm observed for a liquid-crystal phase at 80 degrees C. The quasicrystalline phase coexisted with gel and fluid phase of the pure phospholipid. An analysis of the small-angle x-ray scattering intensity profiles indicated a stoichiometry of one glucosphingolipid per two phospholipid molecules in the complex. Structural transitions monitored in cooling scans by synchrotron x-ray diffraction indicated that a cubic phase transforms initially into a lamellar gel. Thermal studies showed that the gel phase subsequently relaxes into the quasicrystalline phase in an exothermic transition. Electron spin resonance spectroscopy using spin labels located at positions 7, 12, and 16 carbons of phospholipid hydrocarbon chains indicated that order and motional constraints at the 7 and 12 positions were indistinguishable between gel and quasicrystalline phases but there was a significant decrease in order and increase in rate of motion at the 16 position on transformation to the quasicrystalline phase. The results are interpreted as an arrangement of polar groups of the complex in a crystalline array and a quasicrystalline packing of the hydrocarbon chains predicated by packing problems in the bilayer core requiring disordering of the highly asymmetric chains. The possible involvement of quasicrystalline phases in formation of membrane rafts is considered.     

Influence of temperature on complement-dependent immune damage to liposomes

Maximal release of trapped liposomal glucose, in the presence of saturating amounts of liposomal antigen (galactocerebroside), antiserum (anti-galactocerebroside), and complement, was dependent on temperature. At lower temperatures (20--25 degrees C), maximal glucose release was inversely related to liposomal phospholipid fatty acyl chain length (dimyristoyl phosphatidylcholine > dipalmitoyl phosphatidylcholine > distearoyl phosphatidylcholine > sphingomyelin). At higher temperatures (32--35 degrees C) a limiting plateau of glucose release, at approx. 60%, was reached, or approached, by all preparations. Sphingomyelin liposomes still released less glucose than those prepared from other phospholipids, even at 35 degrees C. The titers of antiserum and complement (ABL50/ml and CL50/ml) were dependent on temperature, and differences based on liposomal phospholipid fatty acyl chain length were observed. Analysis of antiserum and complement-dependence on temperature, and on phospholipid type, revealed that although antibody binding to galactocerebroside undoubtedly was subject to steric hindrance due to interference by surrounding phospholipids at 20--25 degrees C, steric hindrance did not play a major role in blocking antibody binding above 32 degrees C.     

Subunit interface mutants of rabbit muscle aldolase form active dimers.

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.     

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.     

An X-ray diffraction study of the effect of alpha-tocopherol on the structure and phase behaviour of bilayers of dimyristoylphosphatidylethanolamine.

The effect of alpha-tocopherol on the thermotropic phase transition behaviour of aqueous dispersions of dimyristoylphosphatidylethanolamine was examined using synchrotron X-ray diffraction methods. The temperature of gel to liquid-crystalline (Lbeta-->Lalpha) phase transition decreases from 49.5 to 44.5 degrees C and temperature range where gel and liquid-crystalline phases coexist increases from 4 to 8 degrees C with increasing concentration of alpha-tocopherol up to 20 mol%. Codispersion of dimyristoylphosphatidylethanolamine containing 2.5 mol% alpha-tocopherol gives similar lamellar diffraction patterns as those of the pure phospholipid both in heating and cooling scans. With 5 mol% alpha-tocopherol in the phospholipid, however, an inverted hexagonal phase is induced which coexists with the lamellar gel phase at temperatures just before transition to liquid-crystalline lamellar phase. The presence of 10 mol% alpha-tocopherol shows a more pronounced inverted hexagonal phase in the lamellar gel phase but, in addition, another non-lamellar phase appears with the lamellar liquid-crystalline phase at higher temperature. This non-lamellar phase coexists with the lamellar liquid-crystalline phase of the pure phospholipid and can be indexed by six diffraction orders to a cubic phase of Pn3m or Pn3 space groups and with a lattice constant of 12.52+/-0.01 nm at 84 degrees C. In mixed aqueous dispersions containing 20 mol% alpha-tocopherol, only inverted hexagonal phase and lamellar phase were observed. The only change seen in the wide-angle scattering region was a transition from sharp symmetrical diffraction peak at 0.43 nm, typical of gel phases, to broad peaks centred at 0.47 nm signifying disordered hydrocarbon chains in all the mixtures examined. Electron density calculations through the lamellar repeat of the gel phase using six orders of reflection indicated no difference in bilayer thickness due to the presence of 10 mol% alpha-tocopherol. The results were interpreted to indicate that alpha-tocopherol is not randomly distributed throughout the phospholipid molecules oriented in bilayer configuration, but it exists either as domains coexisting with gel phase bilayers of pure phospholipid at temperatures lower than Tm or, at higher temperatures, as inverted hexagonal phase consisting of a defined stoichiometry of phospholipid and alpha-tocopherol molecules.     

A new high-temperature transition of crystalline cholesterol in mixtures with phosphatidylserine

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96 degrees C by differential scanning calorimetry (DSC), using a heating scan rate of 2 degrees C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96 degrees C is more rapid at higher incubation temperatures. At 37 degrees C the half-time of conversion is approximately 7 days. Concomitant with the appearance of the 96 degrees C peak the previously known transitions of cholesterol, occurring at approximately 38 degrees C and 75 degrees C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75 degrees C peak takes a longer time than that of the 38 degrees C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75 degrees C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96 degrees C. After several weeks of incubation at 37 degrees C, only the form with a phase transition at 96 degrees C remains. If such a sample undergoes several successive heating and cooling cycles, the 96 degrees C peak disappears and the 38 degrees C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38 degrees C transition that reappears after the melting of the 96 degrees C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96 degrees C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96 degrees C transition, after incubation of the sample for 3 weeks at 37 degrees C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37 degrees C, exhibits a transition at 95 degrees C that reverses on cooling at 44 degrees C, instead of 60 degrees C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96 degrees C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.     

Phase separations of alpha-tocopherol in aqueous dispersions of distearoylphosphatidylethanolamine

The effect of alpha-tocopherol on the structure and thermotropic phase behaviour of distearoylphosphatidylethanolamine was examined by using synchrotron X-ray diffraction methods. There was evidence that alpha-tocopherol does not distribute randomly in the dispersed phospholipid but instead phospholipid phases enriched in alpha-tocopherol are formed. Heating codispersions from lamellar gel phase induced formation of hexagonal-II phase at temperatures below the main transition of the pure phospholipid and which were enriched in alpha-tocopherol. Codispersions containing 5 or 10 mol% alpha-tocopherol were induced to form a cubic phase at temperatures above the lamellar to hexagonal-II phase transition. Such phases were not observed in codispersions containing 2.5 or 20 mol% alpha-tocopherol in which only lamellar and hexagonal-II phases were formed. The space group of the cubic phases were tentatively assigned as Pn3m. Equilibration of codispersions at 4 degrees C results in the formation of lamellar crystalline phases enriched in alpha-tocopherol and phase separated domains of pure phospholipid. Two lamellar crystalline phases were characterized on the basis of their particular wide-angle X-ray scattering patterns. The lamellar crystalline phases were also distinguished from other lamellar phases of the pure phospholipid by the lamellar repeat. Partitioning of alpha-tocopherol into phosphatidylethanolamine domains in membranes may introduce instability into the structure.     

A calorimetric study of the thermotropic behaviour of mixtures of brain cerebrosides with other brain lipids

We have used a computer-controlled differential scanning calorimeter to determine the phases present in mixtures of the brain galactocerebrosides with other representative brain lipids. There are two types of brain galactocerebroside, those which possess an alpha-hydroxy substituent on the acyl chain (HFA) and those that do not (NFA). In the liquid crystalline state both cerebrosides were miscible with all the lipids studied, but in the gel state they were immiscible with cholesterol and the brain phosphatidylcholines. However, cholesterol mixtures in which the cholesterol mole fraction exceeded one third formed homogeneous metastable gel states on cooling from above the melting point of the cerebroside. Relaxation to the stable two phase state took place slowly over several hours. The solubilities of the galactocerebrosides in the other main brain sphingolipid, sphingomyelin, were much higher. Only in the case of the NFA galactocerebroside and at low mole fractions of sphingomyelin was immiscibility detected. Ternary mixtures of the two cerebrosides with sphingomyelin/cholesterol and phosphatidylcholine/cholesterol (PC/Chol) showed different miscibility characteristics. On cooling from 80 degrees C all mixtures formed homogeneous gel states. However, on standing the cerebrosides separated into discrete gel phases in all mixtures but one, that in which HFA galactocerebrosides were mixed with sphingomyelin and cholesterol. The cerebroside in the mixture with the composition closest to that of myelin, HFA/PC/Chol, melted at 38 degrees C. On scanning guinea pig CNS myelin which had been equilibrated at 5 degrees C a transition was detected with Tmax 33 degrees C. On the basis of comparison with the HFA/PC/Chol mixture we propose that the transition in myelin at this temperature is due to the melting of a galactocerebroside gel phase.     

Interaction of spectrin with hemin disaggregates spectrin associations

Crude spectrin preparations were extracted from red cell membranes either in dimeric or tetrameric forms and incubated at 4 degrees C with hemin. The mixtures were subjected immediately or after 18 hours to nondenaturing electrophoresis. It was found that immediately after addition of 0.3 mM hemin, the fraction of spectrin complexed with other skeletal proteins, disaggregated to tetramer and dimer forms. After incubation for 18 hours at 4 degrees C most of the spectrin appeared in two additional bands which contained more hemin and migrated on the gels as molecular weight forms smaller than the dimers. Since SDS electrophoresis showed that spectrin subunits retained their integrity in these mixtures, it was concluded that hemin bound spectrin dissociates with time into monomers. It is suggested that there are pathophysiological implications to the disaggregation of spectrin complexes in the cytoskeleton by hemin.     

X-ray diffraction studies of lipid phase transitions in cholesterol-rich membranes at sub-zero temperatures

In X-ray diffraction studies of hydrated (greater than 60%) cholesterol/dioleoylphosphatidylcholine mixtures the lipid packing band showed an abrupt transition from liquid crystal-type to gel-type position and definition at a temperature which decreased progressively to almost -50 degrees C as the proportion of cholesterol was increased to a saturation level of about 50 mol%. Plots of transition temperature against composition (mol% cholesterol) and of peak position against composition provided evidence of a significant change in phospholipid configuration at about 20 mol% cholesterol. However, the data overall suggested a uniform dispersion of the cholesterol molecules in the phospholipid bilayer at all concentrations up to the saturation point. Parallel studies of hydrated lipid extract of erythrocyte membranes and of several cholesterol-rich membrane preparations showed a similar overall change from liquid crystal-type packing at +20 degrees C to a gel-type packing at -30 degrees C to -40 degrees C but without displaying a defined transition temperature.     

In vivo metabolism of labeled oleic and linoleic acids by the laying hen.

Radioactive oleic and linoleic acids, labeled with 3H in the chain and 14C in the carbonyl group, were administered to white leghorn laying hens. Mixtures fed in separate experiments included: (1) 3H- and 14C-labeled oleic acid, (2) 3H- and 14C-labeled linoleic acid and (3) [3H]oleic aicd and [14C] linoleic acid. The 3H/14C ratios of both the neutral lipid and phospholipid fractions from the egg yolk and of the isolated acids from these lipid fractions were compared to that in the administered mixture. Agreement in the 3H/14C ratios for the neutral lipid fraction from each of the feeding experiments indicated that neither the 3H- and 14C labeled acids nor the oleic or linoleic acids were distinguishable during synthesis of the neutral lipid. Analysis of the phospholipid fractions showed that when dual-labeled mixtures of oleic acid were administered, 3H/14C ratios were elevated and, therefore, there was selective elimination of the 14C label. When dual-labeled mixtures of linoleic acid were administered, the 3H/14C ratios were in agreement; and when the two acids were administered simultaneously as a dual-labeled mixture, there was selective incorporation of linoleic acid. These findings indicate separate metabolic pathways for synthesis of neutral lipid and phospholipid in egg yolk as expected, as well as preferential use of the essential fatty acid in the phospholipid by the hen.