Lipid phase of transverse tubule membranes from skeletal muscle. An electron paramagnetic resonance study.

The lipid phase of transverse tubule membrane was probed with a variety of fatty acid spin labels. The motion of the probe increased as the distance between the spin label and polar head group increased, in agreement with results reported in other membranes. The value of the order parameter at 37 degrees C for a fatty acid spin label containing the label attached to its fifth carbon atom was closer to values reported for bacterial membranes than to the lower values reported for other mammalian membranes. Order parameters for spin labels containing the label nearer to the center of the bilayer were closer to the values reported in other mammalian membranes than to values reported for bacterial membranes. These results indicate that the lipid segments in the vicinity of the polar head group, and less so those near the center of the bilayer, are motionally more restricted in transverse tubules than in other mammalian membranes. In particular, the lipid phase of the transverse tubule membrane is less fluid than that of the sarcoplasmic reticulum membrane. A possible role of the high cholesterol content of transverse tubules in generating the lower fluidity of its lipid phase is discussed.     

Free fatty acids and esters can be immobilized by receptor rich membranes from Torpedo marmorata but not phospholipid acyl chains.

A long chain spin labeled fatty acid and the corresponding ester have been introduced into receptor rich membranes from Torpedo Marmorata. Superimposed to a mobile component, typical of the lipid phase, a strongly immobilized component is seen on the ESR spectra, both at low temperature (?4°C) and at room temperature. An estimation of the amount of immobilized signal as a function of the concentration of spin label in the membrane shows that a saturation is reached which corresponds to approximately twice the concentration of receptor protein. In the same membranes, a spin labeled phosphatidylcholine was introduced by the release of the phosphatidylcholine analog from purified phosphatidylcholine exchange protein, preloaded with this spin label. No immobilized component is seen in this latter case even at low temperatures. Therefore the immobilized component seen with the fatty acid cannot be considered as reporting on an immobilized boundary layer of phospholipids surrounding the proteins. We attribute the immobilized signal seen with fatty acids and esters to a particular interaction of amphiphilic molecules with the cholinergic receptor protein. Very likely this effect can be associated with the local anaesthetic effect detected previously with this fatty acid.     

Correlations between fatty acid distribution in phospholipids and the temperature dependence of membrane physical state

Cytoplasmic membranes of an unsaturated fatty acid auxotroph of Escherichia coli have been studied using spin labeled hydrocarbon probes. These studies reveal that the membrane lipids undergo changes of state at critical temperatures which reflect the physical properties of the fatty acid supplement supplied to the cells during growth. The critical temperatures observed in spin labeled membranes correlate with characteristic temperatures in membrane functions. Lipid analysis reveals that fatty acid composition and distribution in membrane phospholipids are primary determinants of the temperatures at which changes of state are observed in membrane lipids. Fatty acid composition and distribution can also produce unique interactions between certain spin label probes and their lipid environment.     

Influence of membrane-lipid composition on translocation of nascent proteins in heated Escherichia coli

In studies using Escherichia coli we have shown that new protein species appear in the outer membrane fraction with concomitant losses of nascent proteins from the soluble and inner membrane fractions following heat exposure. Of the various explanations for this phenomenon, temperature-induced membrane disorganization appeared the most likely. It is suggested that heat mimics the action of the signal sequence of a protein on the lipid bilayer allowing non-signal-sequence-containing proteins to be translocated. To test this hypothesis we grew E. coli K1060 cells, an unsaturated fatty acid requiring auxotroph, supplemented during growth with fatty acids of varying chain length in an attempt to determine whether biological membranes of varying ability to maintain their bilayer configuration could be constructed. The rationale being that such membranes would allow us to determine whether differences in translocation would occur in cells grown at the same temperature supplemented with either 16:1 or 20:1 unsaturated fatty acids when the cells were subjected to a series of thermal insults. Protein translocation occurred to a greater extent and at lower temperatures in cells supplemented with the longer chain fatty acid. Treatment of outer membranes with either 1 M salt, 6 M urea or high pH and studies determining fluorescent polarization values by scanning up and down through a series of temperatures ranging from 15 to 49 degrees C indicate that the proteins translocated by heat to the outer membrane are integral. Protein translocation may represent an adaptive response to an altered environment enabling the cell to respond to stress by stabilizing its outer membrane.     

Infrared spectroscopic studies of Acholeplasma laidlawaii B membranes. Comparison of the gel to liquid-crystal phase transition in intact cells and isolated membranes

The physical state of the membrane lipids in the plasma membranes of intact, live Acholeplasma laidlawii B cells was probed by Fourier-transform infrared spectroscopy and compared with that in isolated membranes. Infrared spectra of live A. laidlawii B cells, enriched biosynthetically in the presence of avidin, with saturated deuterated and unsaturated non-deuterated fatty acids have been recorded at a variety of temperatures. The results indicate that within the temperature range of the gel to liquid-crystal phase transition, the live cells are able to keep the 'fluidity' of their plasma membranes at a considerably higher value compared to that in the isolated membranes at the same temperature. While this is a generally valid observation, the degree by which live and isolated membranes differ in their liquid-crystal-phase content at a given temperature depends on the nature of the exogenous fatty acid and the temperature of growth.     

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.     

Modulation of the catalytic properties of alpha-chymotrypsin by chemical modification at Tyr 146

XC Sarcoma, Vero and Aedes aegypti plasma membranes have been studied in viable cells and in purified membrane of XC Sarcoma cells by the spin label method. The temperature dependence of the order parameter of fatty acid spin labels is found to be linear in all three cells and membrane and shows no evidence of a lipid phase transition. The order parameter of the fatty acid labels substituted at the 5-position is shown to increase as a function of the cholesterol: phospholipid molar ratio in cells that have been studied to date. Cells attached to their growing surface are studied for the first time by electron paramagnetic resonance spectroscopy (EPR). The resulting spectra are anisotropic due to the non-spherical shape of the cells and show that these labels orient preferentially perpendicular to the cell surface. The viscosity of the extracted XC cell membrane is estimated to be 2.5 P from rotational correlation time measurements of the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO).     

The biosynthetic incorporation of short-chain linear saturated fatty acids by Acholeplasma laidlawii B may suppress cell growth by perturbing membrane lipid polar headgroup distribution

Acholeplasma laidlawii B cells made fatty acid auxotrophic by growth in the presence of the biotin-binding agent avidin grow increasingly poorly at 37 degrees C when supplemented with single exogenous linear saturated fatty acids of decreasing hydrocarbon chain length. Interestingly, this progressive decrease in growth yields with decreasing hydrocarbon chain length is not observed when cells are cultured in the presence of other classes of exogenous fatty acids. Moreover, normal growth is observed is other types of fatty acids with equivalent or shorter hydrocarbon chain lengths, indicating that poor growth in the presence of short-chain linear saturated fatty acids cannot be due to a decrease in membrane lipid bilayer thickness per se. To understand the molecular basis of such growth inhibition, we determined the growth yields, membrane lipid fatty acid and polar headgroups compositions, and phase state and fluidity of the membrane lipids in cells progressively biosynthetically enriched in tridecanoic acid (13:0) or dodecanoic acid (12:0). The growth of fatty acid auxotrophic A. laidlawii B cells grown in the presence of binary combinations of an exogenous fatty acid which supports normal growth on its own and 13:0 or 12:0 revealed that growth inhibition is not observed until 13:0 and 12:0 biosynthetic incorporation levels reach about 90 and 60 mol %, respectively, after which growth is markedly inhibited. Differential scanning calorimetric analyses of membranes from cells maximally enriched in 13:0 indicate that the lipid gel/liquid-crystalline phase transition temperature is unexpectedly high but that at the growth temperature of 37 degrees C, the membrane lipid bilayer is almost exclusively in the liquid-crystalline state but is certainly not excessively fluid. However, high levels of 13:0 incorporation produce a greatly elevated level of the high melting, reversed nonlamellar phase-preferring lipid component monoglucosyl diacylglycerol, and greatly reduced levels of all other membrane lipid components. This marked elevation of monoglucosyl diacylglycerol levels can be rationalized as a regulatory response which maintains the lamellar/nonlamellar phase-forming propensity of the total membrane lipid mixture relatively constant in the face of the biosynthetic incorporation of increasing quantities of short-chain saturated fatty acids, which favor the lamellar phase. However, this lipid biosynthetic response produces a marked decline in the levels of anionic phospholipid and phosphoglycolipid which are probably required to maintain the minimal negative surface charge density of the lipid bilayer, which we suggest is responsible for the observed growth inhibition. This work shows that the lipid biosynthetic regulatory mechanisms present in this organism may sometimes operate at cross purposes such that it is not possible to simultaneously optimize all of the biologically relevant physical properties of the membrane lipid bilayer.     

Magnetic resonance studies of eukaryotic cells. III. Spin labeled fatty acids in the plasma membrane

XC Sarcoma, Vero and Aedes aegypti plasma membranes have been studied in viable cells and in purified membrane of XC Sarcoma cells by the spin label method. The temperature dependence of the order parameter of fatty acid spin labels is found to be linear in all three cells and membrane and shows no evidence of a lipid phase transition. The order parameter of the fatty acid labels substituted at the 5-position is shown to increase as a function of the cholesterol: phospholipid molar ratio in cells that have been studied to date. Cells attached to their growing surface are studied for the first time by electron paramagnetic resonance spectroscopy (EPR). The resulting spectra are anisotropic due to the non-spherical shape of the cells and show that these labels orient preferentially perpendicular to the cell surface. The viscosity of the extracted XC cell membrane is estimated to be 2.5 P from rotational correlation time measurements of the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO).     

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.     

Temperature optimum of insulin-stimulated 2-deoxy-D-glucose uptake in rat adipocytes. Correlation of cellular transport with membrane spin-label and fluorescence-label data.

The effects of temperature alterations between 22 degrees C and 48 degrees C on basal and insulin-stimulated 2-deoxy-D-[1-14C]glucose uptake were examined in isolated rat adipocytes. A distinct optimum was found near physiological temperature for uptake in the presence of maximally effective insulin concentrations where insulin stimulation and hexose uptake were both conducted at each given assay temperature. Basal uptake was only subtly affected. Control and maximally insulin-stimulated cells incubated at 35 degrees C subsequently exhibited minimal temperature-sensitivity of uptake measured between 30 and 43 degrees C. The data are mostly consistent with the concept that insulin-sensitive glucose transporters are, after stimulation by insulin, functionally similar to basal transporters. Adipocyte plasma membranes were labelled with various spin- and fluorescence-label probes in lipid structural studies. The temperature-dependence of the order parameter S calculated from membranes labelled with 5-nitroxide stearate indicated the presence of a lipid phase change at approx. 33 degrees C. Membranes labelled with the fluorescence label 1,6-diphenylhexa-1,3,5-triene, or the cholesterol-like spin label nitroxide cholestane, reveal sharp transitions at lower temperatures. We suggest that a thermotropic lipid phase separation occurs in the adipocyte membrane that may be correlated with the temperature-dependence of hexose transport and insulin action in the intact cells.     

Membranes of Tetrahymena: III. The effect of temperature on membrane core structures and fatty acid composition of Tetrahymena cells

Membrane core structures as revealed by the freeze-etch electron microscopy and the fatty acid composition measured by gas-liquid chromatography have been analyzed in Tetrahymena cells exposed to low temperature for varying periods.When cells were grown to mid-log phase at the optimal growth temperature of 28 °C and then chilled to 10 °C, cell division was inhibited. However, within 16 h the cells adapted to the low temperature.Chilling effected drastic structural alterations in the cores of different membrane types (membranes of the pellicula, the alveolar sacs, the endoplasmic reticulum and the nuclei). In all cases, there was a segregation of smooth faces from particle-rich faces in the fracture planes. However, the native membrane state, i.e. like that of cells grown at 28 °C, reappeared when the cells adapted to the low temperature.The total lipids of Tetrahymena cells contained primarily even-numbered fatty acids ranging from C₁₂ to C₁₈, but we also detected appreciable amounts of C₂₀ acids; this has not been reported before. During the initial phase of chilling, when cell division is inhibited, about 50% of the saturated fatty acids were replaced by unsaturated fatty acids, primarily monoenoic, dienoic and trienoic acids.We conclude that the structural recovery of the membranes in chilled Tetrahymena cells is accomplished by a desaturation of membrane fatty acids. This is discussed with respect to membrane “fluidity”.     

Preparation and properties of vesicles of a purified myelin hydrophobic protein and phospholipid. A spin label study.

Lipophilin, a hydrophobic protein purified from the proteolipid of normal hupid and protein in 2-chloro-ethanol followed by dialysis against buffer. This method resulted in homogeneous incorporation of the protein into lipid vesicles as judged by sedimentation on a sucrose gradient and freeze fracture electreter and the freeze fracture faces contained intramembrane particles. The effect of lipophilin on the organization of the lipid was studied by use of spin label probes. Two distinct components were present in the spectrum of fatty acid spin labels in the lipid-protein vesicles. One was immobilized presumably due to the presence of boundary lipid around the protein and the second component waicles and probably due to a lamellar phase but with a slightly greater order parameter. Lipophilin was found to increase the order parameter linearly with increasing concentration of protein incorporated into the vesicles. However, the phase transition temperature as measured from the 2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPO) solubility parameter was unchanged.     

Effects of growth temperature on protoplast membrane properties in Bacillus megaterium.

Changes in the protoplast membrane of the KM strain of Bacillus megaterium were assessed after growth at 20, 30, or 37 degrees, C. Although the overall membrane concentrations of lipids and proteins were virtually unchanged, increased culture temperature resulted in cells with membranes that contained relatively more unbranched and long-chain fatty acids and more acidic phospholipids, as well as different proportions and numbers of individual proteins. Electrophoretic analysis revealed 23, 31, or 29 protein bands, respectively, in membranes from cells grown at the three temperatures. Protoplasts from cells grown at higher temperatures were considerably less susceptible to lysis by shearing forces. As judged by passive leakage at 30 degrees C, intact cells from cultures grown at 37 degrees C were the least permeable to erythritol. Relatively low ambient concentrations of Ca2+ or Mg2+ protected protoplasts from osmotic lysis but even much higher concentrations left erythritol leakage virtually unaffected. Thus, growth temperature affected not only membrane lipis but also membrane proteins and these changes resulted in membranes with altered mechanical properties and permeabilities.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Effect of Growth Temperature on the Lipid Composition of Cyanidium caldarium: I. Class Separation of Lipids

Cyanidium caldarium was cultured at 20 and 55 C and harvested during exponential growth phase. Comparative lipid studies on each cell type show a decrease by one-half of the total lipid in cells grown at 55 C over cells grown at 20 C. While the distribution of lipid into each of five lipid classes was not influenced by high temperature (55 C), the degree of unsaturation was greatly affected. Ratios of unsaturated to saturated fatty acids in these cells decreased 3-fold with increased temperature in the growth environment. Cells cultured at 20 C contained 30% of their fatty acids as linolenic while this fatty acid could not be detected in cells cultured at 55 C.     

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°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°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°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°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°C, but only 50% at 15°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°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.     

Phase properties of senescing plant membranes. Role of the neutral lipids

Wide-angle X-ray diffraction studies have indicated that rough and smooth microsomal membranes from bean cotyledons acquire increasing proportions of gel phase lipid at physiological temperature as the tissue senesces. In addition, for both types of membrane the lipid phase transition temperature, defined as the highest temperature at which gel phase lipid can be detected, progressively rises with advancing senescence. Liposomes prepared from total lipid extracts of the membranes show a similar increase in transition temperature with age, indicating that separation of the polar lipids into distinct gel and liquid-crystalline domains is not attributable to peculiar protein-lipid interactions. Liposomes prepared from purified phospholipid fractions of the membranes show little change in transition temperature with age, indicating that the altered phase properties of the lipid do not reflect an increase in fatty acid saturation. However, the formation of gel phase lipid that occurs naturally during senescence can be stimulated by preparing liposomes from a mixture of the phospholipid fraction from young membrane and the neutral lipid fraction from old membrane. By adding the separated components of the neutral lipid fraction to purified phospholipid it was found that sterol esters and several unidentified lipids are able to raise the transition temperature of the polar lipids. Sterols have no effect on the phospholipid transition temperature. The data have been interpreted as indicating that several neutral lipids, which presumably increase in abundance with advancing senescence, induce a lateral phase separation of the polar lipids resulting in distinct gel and liquid-crystalline domains of lipid in the senescent membranes.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.