Lipid protein interactions in mitochondria. Spin and fluorescence probe studies on the effect of n-alkanols on phospholipid vesicles and mitochondrial membranes.

The effect of n-butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n-alcohols is related to their hydrophobicity.Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data.As for the spin label data, the effect of the series of aliphatic n-alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.     

SPIN LABEL STUDIES ON THE EFFECT OF ANESTHETICS IN SYNAPTIC MEMBRANES

We have studied the effects of anesthetics on synaptic membranes obtained from pig brain by using stearic acid spin labels. The anesthetics used (butanol, halothane, ketamine) affect the rotational mobility of 16-doxylstearate and the order parameter of 5-doxylstearate. The changes in mobility of 16doxylstearate show a stronger fluidization in the membrane core than in vesicles of lipids extracted therefrom. This effect may be operationally described as a disruption of lipid-protein interactions involving hydrophobic proteins. In fact no disordering is induced on the surface of synaptic membranes as shown by the order of Soioxylstearate, indicating a highly immobilized state of the lipids on the membrane surface. The results are discussed in view of our working hypothesis concerning the role of lipids in modulating protein conformation.     

Molecular mechanism of general anesthesia: II. Spin label studies on synaptic membranes.

In this communication we report the effects of general anesthetics on the mobility and order of spin labeled stearic acid derivatives in synaptic membranes and in bilayers formed from the lipids extracted therefrom. The anesthetics studied abolish the immobilization induced by synaptic membrane proteins on the membrane lipids : this effect, observed particularly in the bilayer core, is interpreted as a labilization of lipid-protein interactions induced by anesthetics.     

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.     

Spin label and microcalorimetric studies of the interaction of DNA with unilamellar phosphatidylcholine liposomes

High-molecular DNA from chicken erythrocytes interacts with 1,2-dipalmitoylphosphatidylcholine in unilamellar liposomes, both in the presence and absence of Mg2+ ions. This interaction results in a phase separation in liposome membranes. The new phase induced by DNA and Mg2+ has a higher gel-liquid crystal phase transition temperature as measured by microcalorimetry. In the liquid crystalline state, the 16- and 5-doxyl stearic acid spin labels indicate changed local bilayer properties at the label position in the new phase.     

Distinct states of lipid mobility in bovine rod outer segment membranes Resolution of spin label results

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0°C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24°C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0°C and a considerable temperature dependence. This component which is not resolved at high temperatures (24–35°C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62–64 G at 0°C, with very little temperature dependence (61–62 G at 35°C), and is attributed to protein aggregation.     

Does propofol alter membrane fluidity at clinically relevant concentrations? An ESR spin label study

General anesthetics have been shown to perturb the membrane properties of excitable tissues. Due to their lipid solubility, anesthetics dissolve in every membrane, penetrate into organelles and interact with numerous cellular structures in multiple ways. Several studies indicate that anesthetics alter membrane fluidity and decrease the phase-transition temperature. However, the required concentrations to induce such effects on the properties of membrane lipids are by far higher than clinically relevant concentrations. In the present study, the fluidizing effect of the anesthetic agent propofol (2,6-diisopropyl phenol: PPF), a general anesthetic extensively used in clinical practice, has been investigated on liposome dimyristoyl-L-alpha phosphatidylcholine (DMPC) and cell (erythrocyte, Neuro-2a) membranes using electron spin resonance spectroscopy (ESR) of nitroxide labeled fatty acid probes (5-, 16-doxyl stearic acid). A clear effect of PPF at concentrations higher than the clinically relevant ones was quantified both in liposome and cell membranes, while no evident fluidity effect was measured at the clinical PPF doses. However, absorption spectroscopy of merocyanine 540 (MC540) clearly indicates a PPF fluidizing capacity in liposome membrane even at these clinical concentrations. PPF may locally influence the structure and dynamics of membrane domains, through the formation of small-scale lipid domains, which would explain the lack of ESR information at low PPF concentrations.     

Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes.

We have developed a saturation transfer EPR (ST-EPR) method to measure selectively the rotational dynamics of those lipids that are motionally restricted by integral membrane proteins and have applied this methodology to measure lipid-protein interactions in native sarcoplasmic reticulum (SR) membranes. This analysis involves the measurement of spectral saturation using a series of six stearic acid spin labels that are labeled with a nitroxide at different carbon atom positions. A large amount of spectral saturation is observed for spin labels in native SR membranes, but not for spin labels in dispersions of extracted SR lipids, implying that the motional properties of those lipids interacting with the Ca-ATPase, i.e., the boundary or annular lipid, can be directly measured without the need for spectral subtraction procedures. A comparison of the motional properties of the restricted lipid, measured by ST-EPR, with those measured by digital subtraction of conventional EPR spectra qualitatively agree, for in both cases the Ca-ATPase restricts the rotational mobility of a population of lipids, whose rotational mobility increases as the nitroxide is positioned toward the center of the bilayer. However, the ability of ST-EPR to directly measure the motionally restricted lipid in a model-independent means provides the greater precision necessary to measure small changes in the rotational dynamics of the lipid at the protein-lipid interface, providing a valuable tool in clarifying the relationship between the physical nature of the protein-lipid interface and membrane function.     

Calcium and chlorpromazine interactions in rat synaptic plasma membranes. A spin-label and fluorescence probe study.

The effects of calcium and of the psychoactive drug chlorpromazine (CPZ) on the rat synaptic plasma membrane have been studied using two stearic nitroxide spin labels having their doxyl groups in positions 5 and 16 and the fluorescent probe 1-anilinonaphtalene-8-sulfonate (ANS). The mobility of the 5-doxyl stearic spin label which probes the membrane phospholipids in the vicinity of their polar heads is decreased in the presence of both compounds. Calcium is more efficient in this respect than CPZ. In spite of this qualitative similarity of action, CPZ inhibits the effect of calcium and vice versa. No modification of the 16-doxyl stearic spectrum has been observed even at high calcium or CPZ concentrations. An increase in fluorescence intensity and a blue shift in the emission wavelength of ANS-probed membranes are observed with very low CPZ concentrations (10^?7 to 10^?5m). With higher concentrations, a further intensity increase and a further blue shift are due to direct interaction between ANS and CPZ. Calcium also increases the fluorescence intensity of ANS-labeled membranes in the concentration range 10^?5–10^?2m. As for the spin-label data, the effects of both compounds are mutually competitive. It is concluded that calcium interacts principally with the phospholipid polar heads of this type of membrane. However, the competition with CPZ suggests indirectly that this ion is also bound to membrane proteins. CPZ has an affinity for membrane lipids only at high concentrations. In its pharmacologically active concentration range, it is located preferentially on the membrane proteins.     

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.     

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

Mechanism of the chilling-induced decrease in proton pumping across the tonoplast of rice cells

The ATP-generated proton pumping across tonoplast vesicles from chilling-sensitive Boro rice (Oryza sativa L. var. Boro) cultured cells was markedly decreased by chilling at 5 degrees C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer was measured by steady-state fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer of the tonoplast vesicles decreased by chilling. The fluidity of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to the decrease in the fluidity of the surface region of the lipid bilayer of the tonoplast, which is caused by the changes in glycolipids.     

Transverse heterogeneity in lipid fluidity in spinach thylakoids,photosystem II preparations,and thylakoid galactolipid vesicles

We have used three doxyl stearic acid spin labels to study the transverse hetero-geneity in lipid fluidity in thylakoids, photosystem II (PS II) preparations, and thylakoid galactolipid vesicles. This comparative study shows that spin labels incorporated into the membrane of the PS II preparation experience far more immobilization than do the same spin labels incorporated into either thylakoids or vesicles prepared from the polar lipids extracted from thylakoids. The spin label immobilization found in the PS II preparation is manifest even near the center of the bilayer, where lipid mobility is normally at its maximum. Analysis of the lipid content of the PS II preparation, relative to chlorophyll, suggests that the PS II preparation may be lipid depleted. This lipid depletion could explain the results presented. However, electron microscopy [Dunahay et al. (1984) Biochim. Biophys. Acta 764:179–193] has not indicated that major delipidation has occurred, and so it remains possible that the immobilization found in the PS II preparation is due primarily to the normal (but close) juxtaposition of adjacent PS II complexes and the cooperative immobilization of their surrounding lipids. Based on the results presented, we conclude that highly mobile lipids are not required for oxygen evolution, the primary photochemistry or the secondary reduction of exogenously added quinones. Unfortunately, the relationship between the plastoquinone pool and the fluidity of the membrane in the PS II preparation remains ambiguous.Abbreviations PS II photosystem II - SDSA 5-doxylstearic acid - 12DSA 12-doxylstearic acid - 16DSA 16-doxylstearic acid - 7N14 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl - chromium oxalate potassium trioxalatochromiate - EPR electron paramagnetic resonance - Chl chlorophyll - MGDG monogalactosyldiacylglycerol - DGDG digalactosyldiacylglycerol     

A spin label study of the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

Electron paramagnetic resonance studies of membrane fluidity in ozone-treated erythrocytes and liposomes

Doxyl stearate spin probes which differed in the attachment of the nitroxide free radical to the fatty acid have been used to study membrane fluidity in ozone-treated bovine erythrocytes and liposomes. Analysis of EPR spectra of spin labels incorporated into lipid bilayer of the erythrocyte membranes indicates an increase in the mobility and decrease in the order of membrane lipids. In isolated erythrocyte membranes (ghosts) the most significant changes were observed for 16-doxylstearic acid. In intact erythrocytes statistically significant were differences for 5-doxylstearic acid. The effect of ozone on liposomes prepared from a lipid extract of erythrocyte lipids was marked in the membrane microenvironment sampled by all spin probes. Ozone apparently leads to alterations of membrane dynamics and structure but does not cause increased rigidity of the membrane.     

Electron spin resonance studies of the effects of lipids on the environment of proteins in mitochondrial membranes

The physical state of mitochondrial membranes has been investigated by means of stearic acid spin labels and of a maleimide spin label covalently bound to protein sulfhydryl groups. Stearic acid spin labels 5-NS and 16-NS show that n-butanol enhances the lipid fluidity of mitochondrial membranes in the whole temperature range between 4 and 37 degrees C; the effects in the hydrophobic membrane core, probed by 16-NS, are already apparent at 10 mM butanol. In liposomes formed of mitochondrial phospholipids, a fluidizing effect appears only at much higher concentration. Such results are compatible with the idea that butanol destabilizes lipid-protein interactions. On the other hand, the ratio between weakly and strongly immobilized SH groups probed by maleimide spin label is only slightly affected in the temperature range of 4-37 degrees C by addition of high concentrations of n-butanol, indicating that the environments probed are stable to agents inducing fluidity changes in the lipids. There are, however, indications that the environment probed by maleimide is affected by lipids, since the spin label, when bound to lipid-depleted mitochondria, becomes more immobilized, reconstitution of such lipid-depleted membranes with phospholipids restores the original spectra.     

Structure of the lipid phase of rauscher murine leukemia virus

The lipid-containing membrane of Rauscher murine leukemia virus was studied using stearic acid spin labels with the nitroxide ring on the C₅ and C₁₆ positions. The environment of the C₅ spin label was found to be much more rigid than that of the C₁₆ spin label. This result, which parallels similar observations in red cell membranes and influenza virus, suggests that the lipid phase of Rauscher murine leukemia virus is arranged in a bilayer.     

The distribution of lipid attached spin probes in bilayers: application to membrane protein topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

Comparison of dynamics of lecithin liposomes and brain total lipid liposomes with synaptosomal membranes. An EPR spectroscopy study.

Dynamics and/or order of the hydrophobic part of phosphatidylcholine (PC) liposomes and rat brain total lipid (TL) liposomes and synaptosomes were studied and compared by EPR spectroscopy using the spin probes 5 or 16-doxyl stearic acid and 14-doxyl phosphatidylcholine. The dynamics and/or order of the hydrophobic part of TL liposomes or synaptosomes were similar but differed largely from those of PC liposomes. The dynamics of the hydrophobic part of the liposomes decreased gradually with the increasing TL/PC ratio in the sample. To obtain in TL liposomes or synaptosomes the same EPR spectrum parameters as in PC liposomes at 37 degrees C, the formers have to be heated to temperatures of approximately 50-60 degrees C. The dynamics and/or order of the hydrophobic part of lecithin liposomes at 5-10 degrees C were comparable with those of TL liposomes or synaptosomes at 37 degrees C. The results emphasize the role of the lipid composition in studies concerning drug-lipid and protein-lipid interactions in model and biological membranes.