Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Effect of growth temperature on the composition of the photosynthetic apparatus in a thermophilic cyanobacterium (Synechococcus sp.)

Abstract A thermophilic strain of Synechococcus sp. was grown under light-limited conditions at its optimum temperature of 58°C and at 38°C in order to investigate the effect of growth temperature on the composition of the photosynthetic apparatus. Cells grown at 38°C had a ratio of Photosystem I to Photosystem II of 2.2, close to that known to occur in unstressed mesophilic strains of Synechococcus . Growth at the higher temperature led to a doubling of the number of Photosystem I reaction centres per cell whilst Photosystem II remained essentially unchanged, causing the Photosystem I/Photosystem II ratio to rise to 4.1. These changes were correlated with an increase in the number of concentric layers of thylakoid membranes from four to nine. It is suggested that these changes result from a higher relative demand for energy (ATP) during growth at elevated temperatures.     

Multiple stages of detergent-erythrocyte membrane interaction--a spin label study

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111).     

Molecular dynamics simulation of dipalmitoylphosphatidylcholine modified with a MTSL nitroxide spin label in a lipid membrane

We investigate the interaction between dipalmitoylphosphatidylcholine (DPPC) and a nitroxide spin label in order to understand its influences on lipid structure and dynamics using molecular dynamics simulations. The system was modified by covalently attaching nitroxide spin labels to the headgroups of two DPPC molecules. (S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate) (MTSL) was used as the spin label. The label position and dynamics were analyzed as was the impact of the modified DPPC on the structure of the surrounding lipids. The modified DPPC molecules locate closer to the center of the membrane than unmodified DPPC molecules. The rotation of the spin label is unrestricted, but there are favored orientations. MTSL depresses the deuterium order parameters of the carbon atoms close to the headgroup in surrounding DPPC molecules. The spin label has no impact on order parameters of carbon atoms at the end of the lipid tails. The lateral diffusion constant of the modified DPPC is indistinguishable from unmodified DPPC molecules. These novel computational results suggest an experimental validation.     

Vesicular stomatitis virus induced membrane changes: A spin label study

Synchronized entry of Vesicular Stomatitis Virus (VSV) into spin labeled cultured human cells resulted in an increase in the rigidity of cell membranes as measured by Electron Spin Resonance Spectroscopy. Treatment of spin labeled cells with homologous interferon alpha did not influence the membrane fluidity, neither did it significantly prevent the VSV induced membrane changes despite its anti-viral protection.     

Cholesterol modulation of lipid-protein interactions in liver microsomal membrane: a spin label study.

ESR spectra of spin probes were used to monitor lipid-protein interactions in native and cholesterol-enriched microsomal membranes. In both systems composite spectra were obtained, one characteristic of bulk bilayer organization and another due to a motionally restricted population, which was ascribed to lipids in a protein microenvironment. Computer spectral subtractions revealed that cholesterol modulates the order/mobility of both populations in opposite ways, i.e., while the lipid bilayer region gives rise to more anisotropic spectra upon cholesterol enrichment, the spectra of the motionally restricted population become indicative of increased mobility and/or decreased order. These events were evidenced by measurement of both effective order parameters and correlation times. The percentages of the motionally restricted component were invariant in native and cholesterol-enriched microsomes. Variable temperature studies also indicated a lack of variation of the percentages of both spectral components, suggesting that the motionally restricted one was not due to protein aggregation. The results correlate well with the effect of cholesterol enrichment on membrane-bound enzyme kinetics and on the behavior of fluorescent probes [Castuma & Brenner (1986) Biochemistry 25, 4733-4738]. Several hypothesis are put forward to explain the molecular mechanism of the cholesterol-induced spectral changes.     

Upper temperature limits for growth and diazotrophy in the thermophilic cyanobacterium HTF Chlorogloeopsis

The effect of temperature and oxygen on diazotrophic growth of the thermophilic cyanobacterium HTF (High Temperature Form) Chlorogloeopsis was investigated using cells grown in light-limited continuous culture at a dilution rate of 0.02 h^-1. Diazotrophy was more sensitive to elevated temperatures than growth with combined nitrogen. The maximum temperature for growth of cultures gassed with CO₂-enriched air was more than 55 °C but less than 60 °C with N₂ as the sole nitrogen source, but between 60°C and 65°C when nitrate was present in the medium. The effect of temperature on nitrogenase activity, photosynthesis and respiration in the dark was determined using cells grown at 55°C. Maximal rates of all three processes were observed at 55°C and rates at 60°C during shortterm incubations were not less than 75% of the maximum. However, nitrogenase activity at 60°C was unstable and decayed at a rate of 2.2 h^-1 under air and at 0.3 h^-1 under argon. Photosynthesis and respiration were more stable at 60°C than anoxic nitrogen fixation. The upper temperature limits for diazotrophic growth thus seem to be set by the stability of nitrogenase.Abbreviations chl chlorophyll a - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - Taps N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid     

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.     

A spin label study of E. coli membrane vesicles

The phase transition in E. coli membrane vesicles has been investigated by the spin labeling technique. N-oxyl-4′,4′-dimethyloxazolidine derivatives of stearic acid were incorporated into the vesicles. The results suggest that there are two phase transitions in these bacterial membrane vesicles (one at ≈20°C and the other at ≈30°C). These two phase transitions may be related to some of the functional properties of the membranes.     

Molecular dynamics of 3,3',5-triiodo-L-thyronine in model membranes: a spin label study

A spin-labeled derivative of 3,3',5-triiodo-L-thyronine, 3-[( alpha-carboxy-4-(4-hydroxy-3-iodophenoxy)-3,5-diiodophenethyl++ +] carbamoyl)-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (SL-T3) has been synthesized. Evaluation of its binding to nuclei after incubation with rat pituitary tumor GH3 cells at 37 degrees C showed that it bound to nuclei with a 18% potency of that of T3. The dynamic interaction of SL-T3 with multilamellar vesicles prepared from dimyristoylphosphatidylcholine (DMPC) was investigated using electron spin resonance techniques. At 31 degrees C, the lateral diffusion constant of SL-T3 in DMPC membranes was found to be 3.0 X 10(-8) cm2/s as determined by the ESR line-broadening method. The temperature dependency of the ESR spectrum of SL-T3 in DMPC multilamellar vesicles showed a break at 23.5 degrees C, which is close to the main phase-transition temperature, 23.7 degrees C, of DMPC membranes. This suggests that the motion of the probe reflects the motion of phospholipids in DMPC membranes, and that the probe itself does not perturb the membrane structure. SL-T3 appears to be a useful probe for studying the motion of thyroid hormone in the plasma membrane of responsive cells.     

The effects of a collagenous extracellular matrix on fibroblast membrane organization : An ESR spin label study

Human skin fibroblasts, both in suspension and cultured within a three-dimensional collagen matrix have been examined by electron spin resonance ESR using the probe 5-doxyl stearic acid. The order of the plasma membrane was found to be strongly influenced by the collagen matrix, being greater for cells within the collagen gel than in suspension. The collagen cultures used in this study were either left attached to the walls of the plastic culture dish (‘attached’ gels) or dislodged and allowed to float freely in the culture medium (‘floating’ gels). Membrane order increased with time in attached gels, reaching a steady value after 2–3 h. A further increase in order was observed when floating gels were prepared 24 h later. Cell morphology within the collagen gel culture was observed to vary considerably, with time and mode of culture. Increased order, over that observed in suspension, was also found for cells attached to other substrata. The data indicate that the increase in membrane order observed in cells embedded within a three-dimensional collagen gel matrix compared with cells in suspension does not correlate with a particular cell morphology in the gel, but rather appears to result from the establishment of adhesive interactions with the surrounding collagen fibres.     

Photosynthetic activities of a membrane preparation of the thermophilic cyanobacterium Mastigocladus laminosus

Photosynthetically active membranes have been prepared from the thermophilic cyanobacterium Mastigogladus laminosus by treatment with lysozyme. The membranes were active in electron transport through photosystem I and II as well as in photophosphorylation and proton uptake. Cells were grown at 40°, 45° and 55°C respectively. The temperature optimum of oxygen evolution of whole cells was about 10°C higher than the growth temperature. In isolated membranes the temperature optimum for cyclic photophosphorylation was identical to the growth temperature of the cells whereas the optimum for photosystem II electron transport never exceeded 40°C. Photophosphorylation was inhibited by N, N-dicyclohexyl carbodiimide (DCCD), carbonyl-cyanide-m-chlorophenylhydrazone and NH₄Cl, whereas proton uptake was enhanced by DCCD. Electron transport was slightly inhibited by these treatments. The membranes could be stored for several weeks at-20°C in 50% glycerol without any loss in the activities.Abbreviations DPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - PMS N-methylphenazonium methosulfate - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - TMP 20 mM Tris-HCl buffer pH 7.8, 10 mM MgCl₂, 5 mM phosphate buffer pH 7.8     

Effect of calcium, insulin and growth hormone on membrane fluidity. A spin label study of rat adipocyte and human erythrocyte ghosts

ESR spectra were recorded from rat epididymal adipocyte ghosts labeled with the 5-nitroxide stearic acid spin probe, I(12,3). Polarity-corrected and approximate order parameters, that are sensitive to the flexibility of the incorporated label, were used to evaluate the membrane lipid fluidity. Addition of CaCl2 a 37 degrees C decreased the fluidity, as indicated by positive increases in the order parameters. The ordering effect of Ca2+ was concentration-dependent, reached saturation at approx. 3--4 mM, and was completely reversed by excess EGTA. Previous studies indicated that low- and high-affinity sites on adipocyte plasma membranes are able to bind 45Ca2+, and our results suggest that Ca2+-induced alterations in the lipid fluidity involve cation binding to low-affinity sites. The cellular movements of Ca2+ and, in particular, the binding of Ca2+ to the plasma membrane may play important roles in insulin's action on fat cell function. The possibility that insulin directly alters the membrane fluidity was tested by adding hormone to freshly-prepared I(12,3)-labeled adipocyte ghosts. Insulin, at concentrations (10(-6) M) that enhance glucose uptake into intact adipocytes, did not affect the fluidity of ghosts suspended in buffers with or without Ca2+. The fluidities of I(12,3)-labeled rat adipocyte ghosts or human erythrocyte ghosts were also unaffected by various forms of human growth hormone.     

Electron spin resonance spin label studies of plasma fibronectin: effect of temperature

Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin.     

Cytochrome c interaction with the mitochondrial membrane: A spin label study

Horse-heart ferrocytochrome c has been labeled with N-(2,2,5,5-tetramethyl-3-pyrrolidinyl-1-oxyl) iodoacetamide at methionine-65. The paramagnetic resonance spectrum of labeled ferricytochrome c indicates a weak immobilization of the radical (τ_c = 9.3·10⁻¹⁰ sec) which becomes stronger upon binding of labeled cytochrome c to cytochrome c-depleted mitochondrial membranes (τ_c = 3.3·10⁻⁹ sec). The hyperfine coupling constant remains, however, unchanged (16.7 ± 0.1 gauss) indicating that the cytochrome c binding site is highly polar. The region where cytochrome c is bound to the membrane is insensitive to large variations of medium viscosity.     

Adamantyl nitroxide: A spin label for probing membrane surfaces

In an effort to understand more about the perturbing properties of adamantane-like molecules on biological membranes, the spin probe adamantyl nitroxide (2,2′-dimethyl-5-adamantyl oxazolidine-N-oxyl) was synthesized, purified and characterized. Electron paramagnetic resonance (EPR) spectra were then obtained from 1:50 and 1:200 mixtures of adamantyl nitroxide with dipalmitoyl and dipalmityl phosphatidylcholine multibilayers. Above the phase transition temperature of these lipids (41°C for dipalmitoyl phosphatidylcholine and 43°C for dipalmityl pholphatidylcholine) the spectra of adamantyl nitroxide are similar to control spectra obtained in liquid oleic acid. Below the phase transition temperatures, however, spectral differences were observed depending on: (1) the concentration of the spin probe in the lipid; (2) the linkage between the polar head group and the hydrocarbon tails of the phospholipid; (3) the temperature of the sample. Partitioning of adamantyl nitroxide between the aqueous and hydrocarbon phases of the sample is most prominent at probe-to-lipid ratios of 1:200 and at temperatures below the pre-transition temperature of the lipid (around 33°C). Computer simulations of the above results, as well as additional experiments performed at 35 GHz, show that the results arise from true partitioning and not from asymmetric probe motion.Two conclusive results of these experiments are that spectra of adamantyl nitroxide in phospholipid multibilayers are sensitive to probe concentration and to the physical characteristics of the phospholipid which they probe. The spectral differences which arise when adamantyl nitroxide is used with ether- and ester-linked phospholipids indicate that it is a sensitive probe of membrane surfaces. Employment of this molecule in membrane research should prove to be useful in obtaining additional information about membrane surface events.