Analysis of Dictyostelium discoideum plasma membrane fluidity by electron spin resonance.

Dictyostelium discoideum grown axenically in media containing polyunsaturated fatty acids exhibited normal growth rates but impaired differentiation (Weeks, G. (1976) Biochim. Biophys. Acta 450, 21--32). Since cell-cell contact is vital for differentiation but unnecessary for growth we have examined the isolated plasma membranes of these cells. The lipids of the plasma membranes of cells grown in the presence of polyunsaturated fatty acids contain considerable quantities of these acids, but the total phospholipid and sterol contents of the plasma membrane are close to normal. Electron spin resonance studies using 5-doxyl-stearic acid as the spin probe reveal two things. Firstly, there are no detectable characteristic transition temperatures in the plasma membranes of D. discoideum. Secondly, the plasma membranes of cell grown in the presence of polyunsaturated fatty acids have essentially the same fluidity as that of the control cells. The possible significance of this result to impaired cell-cell interaction is discussed.     

Radiation-induced lipid peroxidation and the fluidity of erythrocyte membrane lipids

The effect of radiation-induced peroxidation on the fluidity of the phospholipids of the erythrocyte membrane was studied using both erythrocyte ghosts and liposomes formed from the polar lipids of erythrocytes. In liposomes, the oxidation of the phospholipids increased with radiation dose, but there was no change in the fluidity of the lipids as measured by spin-label motion. Under the same conditions of irradiation, no oxidation of phospholipid was detected in erythrocyte ghosts, although changes occurred in the motion of spin labels intercalated with the membrane. These changes were attributed to radiation-induced alterations in the membrane proteins. It is concluded that alterations in motion of spin labels, observed with intact membranes after irradiation, are most likely the result of changes in the structure of membrane proteins rather than the lipids.     

The fluidity and organization of mitochondrial membrane lipids of the brown adipose tissue of cold-adapted rats and hamsters as determined by nitroxide spin probes.

A detailed study of lipid fluidity and organization in the mitochondria of the brown adipose tissue from warm- and cold-adapted rats (nonhibernators) and hamsters (hibernators) is made in order to delineate any relationship between lipid properties and the ability to lower body temperature after cold-adaptation. Complete phospholipid analyses are presented; the data are very similar for cold- and warm-adapted rats, and for cold- and warm-adapted hamsters, but the rat lipids have a higher degree of unsaturation than those of the hamsters. Spin probe analogs of stearic acid and cholestane were used to investigate at the molecular level the fluidity and order of the mitochondrial lipids. Studies were made on intact mitochondria, and in liposomes and oriented multibilayers of extracted lipids. In no case was evidence found for a phase transition in the lipids, or for a relationship between the lipid fluidity in brown adipose tissue mitochondria and the ability to survive at lowered body temperatures. The spin probes generally had a decreased mobility in mitochondria relative to extracted lipids. The electron spin resonance spectra were analyzed to include order- and time-dependent phenomena by a recent stochastic method. The results show that more approximate analyses for order parameters and correlation times can yield incorrect conclusions. As segmental motion decreases in rate, order parameters will be overestimated. Decreasing rates of pseudoisotropic motion lead to incorrect estimates of rotational correlation times. Either of the above can result in the inference of an artifactual phase transition in the lipids.     

Applications of electron spin resonance spectroscopy to the identification of radicals produced during lipid peroxidation

Electron spin resonance (ESR) spectroscopy, which is the only commonly available method for directly detecting free radicals in biological systems, has now been quite extensively used to study radicals produced by metabolism of xenobiotic chemicals and the interaction of such species with lipid molecules. This review examines a variety of different xenobiotic systems and tissues and summarises the information obtained from these studies, with particular reference to the elucidation of the nature of the radicals involved in the initiation and propagation of lipid peroxidation.     

Conformational transition of polypeptide chain elongation factor G as determined by electron spin resonance.

The conformational transition of the polypeptide chain elongation factor G (EF-G) induced by interaction with guanine nucleotide has been investigated by means of the spin-labeling technique. Various spin-label probes were attached specifically to the sulfhydryl group of the protein that is essential for binding to ribosomes, and the effects of these ligands on the electron spin resonance (ESR) spectra were examined. It was found that the ESR spectra of EF-G labeled with nitroxide maleimide reagents were modified by the addition of various guanine nucleotides such as GDP, GTP and, to a lesser extent, by Gpp(NH)p and Gpp(CH2)p, indicating that conformational changes accompany the binding of nucleotide ligand. However, the ESR spectra of labeled EF-G-GDP and EF-G-GTP were almost identical. On the other hand, when EF-G was labeled with nitroxide iodoacetamide reagents, a clear difference in the ESR spectra of EF-G-GDP and EF-G-GTP derivatives was observed. In this case, the spectral shape of the spin-labeled EF-G in the presence of GTP or its analogs, Gpp(NH)p or Gpp(CH2)p, was quite similar to that of free, unliganded EF-G derivative. These results, together with those previously obtained using hydrophobic probes (Arai, Arai, & Kaziro (1975) J. Biochem. 78, 243-246) demonstrate the existence of an EF-G-guanine nucleotide binary complex. They also indicate that there is a substantial difference in conformation between free EF-G, EF-G-GDP, and EF-G-GTP near the active site essential for interaction with ribosomes.     

Sulfite-induced lipid peroxidation in chloroplasts as determined by ethane production

Ethane formation, as a measure of lipid peroxidation, was studied in spinach (Spinacia oleracea L.) chloroplasts exposed to sulfite. Ethane formation required sulfite and light, and occurred with concomitant oxidation of sulfite to sulfate. In the dark, both ethane formation and sulfite oxidation were inhibited. Ethane formation was stimulated by ferric or ferrous ions and inhibited by ethylenediamine tetraacetate. The photosynthetic electron transport modulators, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and phenazine methosulfate, inhibited both sulfite oxidation and ethane formation. Methyl viologen greatly stimulated ethane formation, but had little effect on sulfite oxidation. Methyl viologen, in the absence of sulfite, caused only a small amount of ethane formation in comparison to that produced with sulfite alone. Sulfite oxidation and ethane formation were effectively inhibited by the radical scavengers, 1,2-dihydroxybenzene-3,5-disulfonic acid and ascorbate. Ethanol, a hydroxyl radical scavenger, inhibited ethane formation only to a small degree; formate, which converts hydroxyl radical to superoxide radical, caused a small stimulation in both sulfite oxidation and ethane formation. Superoxide dismutase inhibited ethane formation by 50% when added at a concentration equivalent to that of the endogenous activity. Singlet oxygen did not appear to play a role in ethane formation, inasmuch as the singlet oxygen scavengers, sodium azide and 1,4-diazobicyclo-[2,2,2]-octane, were not inhibitory. These data are consistent with the view that O₂ is reduced by the photosynthetic electron transport system to superoxide anion, which in turn initiates the free radical oxidation of sulfite, and the free radicals produced during sulfite oxidation were responsible for the peroxidation of membrane lipids, resulting in the formation of ethane.     

Order and fluidity of lipid membranes as determined by fluorescence anisotropy decay

The fluorescence anisotropy decay of four different probes in bilayers of dimyristoylphosphatidylcholine was measured. The probes are diphenylhexatriene, diphenyloctatetraene, trimethylaminodiphenylhexatriene, and trans-parinaric acid. The data for each probe were analyzed in terms of two orientational order parameters, the ordinary order parameter and a higher one, and two rotational diffusion coefficients. The order parameters are largely independent of probe size, but depend on the position of the probes along the membrane normal, thus reflecting the profile of lipid order. If a probe is located in the plateau region of lipid order, its order parameters are interpreted as representing the rigid-body order of lipids. According to this interpretation, the total lipid order in the plateau region originates about equally from rigid-body order and conformational order. The two order parameters obtained for each probe are used to derive approximate angular distributions of the probe molecules. The diffusion coefficient for rotation about the long molecular axis is found to be infinitely large, indicating unhindered rotation about this axis. The diffusion coefficient for rotation about the short molecular axes is evaluated for a viscosity which results as 0.2 poise. This viscosity for rotational diffusion is an order of magnitude smaller than the viscosity for lateral diffusion indicating that at least two viscosities are required to characterize the fluidity of a lipid membrane.Abbreviations FAD fluorescence anisotropy decay - DMR deuterium magnetic resonance - ESR electron spin resonance - DMPC dimyristoylphosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DPO 1,6-diphenyl-1,3,5,7-octatetraene - TMA-DPH 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene - tPnA trans-parinaric acid - NPN N-phenyl-1-naphthylamine - BBO 2,5-bis(4-biphenylyl)oxazole     

Antioxidant capacity in rat skeletal muscle tissues determined by electron spin resonance

The amount of radical scavenging activity in muscle is unknown. The present study examines whether electron spin resonance (ESR) could measure and distinguish antioxidant capacity in muscle with different contractile and metabolic characteristics. Specimens of the soleus, plantaris, gastrocnemius (deep/surface portions), heart and diaphragm were obtained from female Wistar rats (n=7; 12 weeks old). Scavenging activity against superoxide anions in these specimens were determined by ESR using a spin-trapping chemical (5,5-dimethyl-1-pyrroline-N-oxide). The ESR signal intensity of reaction mixtures containing muscle tissues was significantly lower in the heart, soleus, diaphragm and deep portion of the gastrocnemius than in the plataris and surface portion of the gastrocnemius. Thus, the amount of scavenging activity converted into superoxide dismutase activity was the highest in the heart, and higher in the soleus, diaphragm and deep portion of the gastrocnemius than in other muscles (ANOVA, P<0.01). In addition, scavenging activity significantly correlated with citrate synthase activity (r=0.72, P<0.01, n=42) and myoglobin content (r=0.63, P<0.01, n=42). These findings suggested that ESR and spin-trapping can be detect differences in free radical scavenging activity among muscle tissues with different metabolic characteristics.     

Altered cellular membrane fluidity levels and lipid peroxidation during experimental pancreas transplantation

Although the pathogenesis of ischemia reperfusion (IR) injury is based on complex mechanisms, free radicals play a central role. We evaluated membrane fluidity and lipid peroxidation during pancreas transplantation (PT) performed in 12 pigs (six donors and six recipients). Fluidity was measured by fluorescence spectroscopy, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations were used as an index of lipid oxidation. Pancreatic tissues were collected as follows: (A) donor, immediately before vascular clamping; (B) graft, following perfusion lavage with University of Wisconsin preservation fluid; (C) graft, after 16?h of cold ischemia; and (D) recipient, 30?min vascular postreperfusion. Fluidity and MDA and 4-HDA concentrations were similar in cases A, B, and C. However, there was significant membrane rigidity and increased lipid peroxidation after reperfusion (D). These findings suggest that reperfusion exaggerates oxidative damage and may account for the rigidity in the membranes of allografts during PT.     

Perturbations to lipid bilayers by spectroscopic probes as determined by dielectric measurements

Dielectric measurements on lecithin/cholesterol bimolecular lipid membranes have indicated that the series of extrinsic fluorescent probe molecules, the $\text{n-(9-}\text{anthroyloxy}\text{)}$ fatty acids, cause significant perturbation to the bilayer structure at concentrations equivalent to those used in fluorescence experiments (0.1 mol%). Perturbations were observed in the capacitance and conductance of the electrically distinct substructural regions of the bilayer that were consistent with the putative location of the probe molecules. Inclusion of stearic acid decreased the thickness of the hydrocarbon region of the membrane, presumably by expanding the average surface area per unit membrane mass, and also significantly disrupted the surface regions. The attachment of the anthroyloxy moiety to position 2 of a fatty acid accentuated both these effects. Attachment at position 12 had the reverse effect by increasing the volume of the hydrocarbon region without further disturbance of the surface organisation. The 9-positioned probe had an intermediate effect. The degree of perturbation by the 2-positioned probe was dependent on the probe concentration within the range (probe:lipid) 1:1000 to 1:10 000. The technique therefore detects perturbation of structure at probe levels which are lower than those commonly used in fluorescence-labelling experiments.     

Factors altering the membrane fluidity of spinach thylakoid as determined by fluorescence polarization

Alterations in fluidity of thylakoid membranes isolated from spinach chloroplasts in response to sodium bisulfite (NaHSO₃), hydrogen peroxide (H₂O₂), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and free linoleic acid (LA) were investigated by means of a fluorescence polarization study with 1,6-diphenyl-1,3,5-hexatriene as the fluorescence probe. A decrease in fluidity and an increase in microviscosity of membrane were caused by NaHSO₃ and H₂O₂ treatment. In contrast, SDS and BSA were found to increase thylakoid membranes fluidity and decrease microviscosity, in which the corresponding correlation coefficients were −0.9995 to −0.9516 (SDS) and −0.9359 (BSA), respectively. No changes in thylakoid membranes fluidity induced by free LA were found until its concentration above 5 mM where the polarization value (P value) declined (increased fluidity). The results suggest that the changes in thylakoids membrane fluidity might depend on the characteristics, mechanism and extent of the interactions between membrane components and compounds added.     

Effects of intramembrane particle aggregation on erythrocyte membrane fluidity: an electron spin resonance study in normal and in dystrophic subjects

Mobilization and aggregation of intramembrane particles (IMPs) are physiological events observed in various cells. In erythrocyte membranes, aggregation of IMPs can be induced by the exposure of partially desprectrinized erythrocyte membranes to acidic pH. We investigated the association between IMPs aggregation, protein mobility, and membrane fluidity in erythrocyte membranes of healthy controls and Duchenne muscular dystrophy (DMD) patients by using electron spin resonance and specific spin labels for membrane proteins and lipids. In erythrocyte membranes of control subjects, the partial spectrin removal induced a decreased segmental motion of protein spin label indicating an increase of protein-protein interactions. Stearic acid spin labels 5- and 16-(N-oxyl-4,4'-dimethyloxazolidine) showed that the treatment induces an increase of membrane fluidity. In DMD patients, both treated and untreated erythrocyte membranes showed changes of membrane fluidity when compared to those of the controls. Our results suggest that defects in the interactions between skeletal proteins and/or between membrane and skeleton components may contribute to the alterations of erythrocyte membranes in DMD.     

Concentration dependence of nitroxyl spin probes in liposomal solution: electron spin resonance and overhauser-enhanced magnetic resonance studies

In this work, the detailed studies of electron spin resonance (ESR) and overhauser-enhanced magnetic resonance imaging (OMRI) were carried out for permeable nitroxyl spin probe, MC-PROXYL as a function of agent concentration in liposomal solution. In order to compare the impermeable nature of nitroxyl radical, the study was also carried out only at 2?mM concentration of carboxy-PROXYL. The ESR parameters were estimated using L-band and 300?MHz ESR spectrometers. The line width broadening was measured as a function of agent concentration in liposomal solution. The estimated rotational correlation time is proportional to the agent concentration, which indicates that less mobile nature of nitroxyl spin probe in liposomal solution. The partition parameter and permeability values indicate that the diffusion of nitroxyl spin probe distribution into the lipid phase is maximum at 2?mM concentration of MC-PROXYL. The dynamic nuclear polarization (DNP) parameters such as DNP factor, longitudinal relaxivity, saturation parameter, leakage factor and coupling factor were estimated for 2?mM MC-PROXYL in 400?mM liposomal dispersion. The spin lattice relaxation time was shortened in liposomal solution, which leads to the high relaxivity. Reduction in coupling factor is due to less interaction between the electron and nuclear spins, which causes the reduction in enhancement. The leakage factor increases with increasing agent concentration. The increase in DNP enhancement was significant up to 2?mM in liposomal solution. These results paves the way for choosing optimum agent concentration and OMRI scan parameters used in intra and extra membrane water by loading the liposome vesicles with a lipid permeable nitroxyl spin probes in OMRI experiments.     

Dynamic molecular structure of DPPC-DLPC-cholesterol ternary lipid system by spin-label electron spin resonance

The hydrated ternary lamellar lipid mixture of dipalmitoyl-PC/dilauroyl-PC/cholesterol (DPPC/DLPC/Chol) has been studied by electron spin resonance (ESR) to reveal the dynamic structure on a molecular level of the different phases that exist and coexist over virtually the full range of composition. The spectra for more than 100 different compositions at room temperature were analyzed by nonlinear least-squares fitting to provide the rotational diffusion rates and order parameters of the end-chain labeled phospholipid 16-PC. The ESR spectra exhibit substantial variation as a function of composition, even though the respective phases generally differ rather modestly from each other. The Lalpha and Lbeta phases are clearly distinguished, with the former exhibiting substantially lower ordering and greater motional rates, whereas the well-defined Lo phase exhibits the greatest ordering and relatively fast motional rates. Typically, smaller variations occur within a given phase. The ESR spectral analysis also yields phase boundaries and coexistence regions which are found to be consistent with previous results from fluorescence methods, although new features are found. Phase coexistence regions were in some cases confirmed by observing the existence of isosbestic points in the absorption mode ESR spectra from the phases. The dynamic structural properties of the DPPC-rich Lbeta and DLPC-rich Lalpha phases, within their two-phase coexistence region do not change with composition along a tie-line, but the ratio of the two phases follows the lever rule in accordance with thermodynamic principles. The analysis shows that 16-PC spin-label partitions nearly equally between the Lalpha and Lbeta phases, making it a useful probe for studying such coexisting phases. Extensive study of two-phase coexistence regions requires the determination of tie-lines, which were approximated in this study. However, a method is suggested to accurately determine the tie-lines by ESR.     

Alterations in erythrocyte membrane fluidity by phenylhydrazine-induced peroxidation of lipids

The addition of phenylhydrazine (.05 – 0.5 mM) to hemoglobin-free human erythrocyte membranes results in the peroxidation of endogenous phospholipids as measured by the thiobarbaturic acid reaction. The incorporation of 1,6-diphenyl-1,3-5-hexatriene into these membranes revealed a decrease in bulk lipid fluidity. Additionally, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate was decreased and red-shifted after phenylhydrazine treatment of the membranes. The results obtained are consistent with the view that changes in the physical state of plasma membranes subsequent to the peroxidation of membrane lipids may be a determinant of the mechanical properties of drug-treated as well as aging cells.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

The effect of ACTH on rat brain synaptic plasma membrane lipid fluidity

The effect of ACTH on the lipid fluidity was examined in synaptic plasma membranes from rat forebrain. ACTH1-24 increased the fluidity of the synaptic plasma membranes in a dose-dependent way, the lowest effective dose being 10(-5) M. The shorter N-terminal fragment ACTH1-10 was not effective. The significance of this finding is discussed in relation to the known effects of ACTH on synaptic membrane phosphorylation.     

Endogenous proteolysis of the human erythrocyte membrane as studied by two-dimensional gel electrophoresis and electron spin resonance

1. Endogenous proteolysis in human erythrocyte membranes was studied in human erythrocyte membranes incubated at 37 degrees C by monitoring changes in 2-D electrophoretic pattern of membrane polypeptides and in the spectra of maleimide-spin labeled membranes. 2. A strong effect of exogenous proteases derived from contaminating other blood elements was found, resulting in formation of specific spots on 2-D electropherograms, requiring very careful leukocyte removal in investigations of red cell membrane protein composition and proteolysis. 3. Studies of the effects of protease inhibitors and Ca2+ confirmed a complex pattern of endogenous red cell membrane proteolysis ("self-digestion") involving many substrates and enzymes. 4. A promoting effect of high concentrations (150 mM) of Ca2+ on endogenous red cell membrane proteolysis was found.