A spin-label study of the viscosity profile of sarcoplasmic reticular vesicles.

Sarcoplasmic reticular vesicles were prepared from both lobster and rabbit muscle. A variety of water-soluble, lipid-soluble, and alkylating spin labels were used to treat the sarcoplasmic reticular vesicles. All spin label analyses were carried out with and without NiCl₂. Nickel is used to remove spin label signal originating from outside of, on the surface of, or localized in the outermost part of the outer bilayer half of the sarcoplasmic reticular membrane.We conclude that the hydrocarbon portion of sarcoplasmic reticular vesicles has symmetry in regard to the physical properties that limit spin label motion; however, we find that the membrane interface limits spin label motion more on the inner surface than the outer surface and that the trapped aqueous volume which is sampled by water soluble spin labels inside the vesicle enclosure limits spin label motion much more than the average aqueous medium outside.     

Spin-Labeled Neurospora Mitochondria

Spin-label studies were carried out on Neurospora mitochondria under in vivo and in vitro labeling conditions. A long-chained spin-labeled fatty acid was incorporated by Neurospora and was found in mitochondrial phospholipids. The molecular motion at various temperatures was different from that for the same spin label under in vitro labeling conditions. The results for spin-labeled mitochondria were compared with those from isolated lipids and with those from aggregates of spin-labeled fatty acid and isolated bovine serum albumin. These comparisons suggest that the hydrocarbon portions of membranes are relatively fluid and are not extensively restricted in motion by association with proteins.     

Localization of spin labels in oat leaf protoplasts

An assay based on light-mediated oxidation was used to determine whether specific spin labels were partitioned throughout the protoplast or retained in the plasmalemma of Avena sativa L. cv. Garry and Park. Many classes of spin label were tested, including phospholipids, fatty acid, fatty acid methyl ester, maleimide, iodoacetamide, short chain hydrocarbon, androstane, 2,2,6,6-tetramethyl-4-aminopiperidinooxyl (TEMPAMINE) and 2,2,6,6-tetramethylpiperidinooxyl (TEMPO). All except the phosphotidylcholine spin label were found to partition throughout the cell. The phosphotidylcholine spin label may have been selectively retained in the plasmalemma.     

Effect of various sterols on the 22Na permeability and fluidity of phospholipid bilayer membranes.

Sodium-22 efflux was measured in multilamellar liposomes composed of egg lecithin, dicetylphosphate, and various sterols. In a parallel series of experiments a spin labelled fatty acid ester was incorporated into similar vesicles and the molecular motion of the spin label monitored by electron spin resonance spectroscopy. Spin lable mobility was used as a measure of phospholipid hydrocarbon chain motion. There was a poor correlation between the effects of these sterols on sodium permeability and their effects on the motion of the lipid chains. It is postulated that sterols alter sodium transport not only through a reduction in the motional freedom of membrane lipids, but also through changes in the partitioning of sodium between membrane and aqueous phases.     

Spin Label Motion in Fatty Acids

Spin labels dissolved in highly purified fatty acid systems exhibit nearly identical tumbling rates in liquid and solid phases. Even though the spin labels do not have the same molecular geometry as the lipid matrix the melting point of the matrix can be inferred by measurements of the temperature dependency of molecular motion.     

A method to evaluate the antioxidant system for radicals in erythrocyte membranes

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.     

Relation of unsaturated fatty acid composition of rat liver mitochondria to oscillation period, spin label motion, permeability and oxidative phosphorylation

An investigation was made of the influence of mitochondrial unsaturated fatty acid composition on the following mitochondrial parameters: oscillation period, spin label motion (ESR), permeability, and oxidative phosphorylation. Liver mitochondria from rats fed diets deficient in or supplemented with essential fatty acids showed approximately the same total number of unsaturated fatty acids but changed unsaturation levels. Electron microscopy showed that the morphology of the inner membrane compartment was unchanged.

Two differences were correlated with unsaturated fatty acid composition: (1) a slower frequency (or time period) of the oscillatory state of energy-dependent ion transport and (2) a reduction in the motional freedom of each of three spin labels (12NS, 5N10 and 7N14). The increase in oscillation period could arise from a number of rate-limiting processes, including permeability of mitochondria to various anions, cations, and substrate metabolites. However, when the permeability of mitochondria to such substances was tested, no changes were observed in passive or active uptake of these substances or in the efficiency of oxidative phosphorylation under steady-state conditions.

Thus, the two parameters, oscillation period and freedom of spin label motion, which are dependent upon large domains of the mitochondrial membranes, are significantly influenced by the change in unsaturated fatty acid composition in essential fatty acid-depleted mitochondria, even though processes such as permeability of ionic materials and oxidative phosphorylation were not measurably affected by these changes in unsaturated fatty acid composition.     

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).     

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.     

Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein

Nitroxide spin labels were incorporated into selected sites within the β-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Cα carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.     

Rotational dynamics of protein and boundary lipid in sarcoplasmic reticulum membrane

We have used spin labels and electron paramagnetic resonance (EPR) to study the correlation between the rotational dynamics of protein and lipid in sarcoplasmic reticulum (SR) membranes. A short-chain maleimide spin label was used to monitor the submillisecond rotational mobility of the Ca-ATPase enzyme (using saturation transfer EPR); a free fatty acid spin label was used to monitor the submicrosecond rotational mobility of the bulk lipid hydrocarbon chains (using conventional EPR); and a fatty acid spin label derivative (long-chain maleimide) attached to the enzyme was used to monitor the mobility of hydrocarbon chains adjacent to the protein (i.e., boundary lipid). In the native SR membranes, the protein was highly mobile (effective correlation time 50 microseconds). The spectra of the hydrocarbon probes both contained at least two components. For the unattached probe, the major component indicated nearly as much mobility as in the absence of protein (effective rotational correlation time 3 ns), while a minor component, corresponding to 25-30% of the total signal, indicated strong immobilization (effective correlation time greater than or equal to 10 ns). For the attached hydrocarbon probe, the major component (approximately 70% of the total) was strongly immobilized, and the mobile component was less mobile than that of the unattached probe. When the lipid-to-protein ratio was reduced 55% by treatment with deoxycholate, protein mobility decreased considerably, suggesting protein aggregation. A concomitant increase was observed in the fraction of immobilized spin labels for both the free and attached hydrocarbon probes. The observed hydrocarbon immobilization probably arises in part from immobilization at the protein-lipid boundary, but protein-protein interactions that trap hydrocarbon chains may also contribute. When protein aggregation was induced by glutaraldehyde crosslinking, submillisecond protein mobility was eliminated, but there was no effect on either hydrocarbon probe. Thus protein aggregation does not necessarily cause hydrocarbon chain immobilization.     

Motion of spin-labeled fatty acids in murine macrophages. Relation to cellular phagocytic activity.

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels. Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings (2 T11) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives. Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 degrees C, or by fixing them with 0.25% glutaraldehyde. No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with cis- or trans-unsaturated fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

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).     

Transverse motion of chlorophyll derivatives in phospholipid bilayers

Chlorophyll derivatives were synthesized with spin labels attached to the porphyrin ring. These labels were incorporated into egg phosphatidylcholine vesicles in order to estimate the transbilayer motion (flip-flop) of this class of photosynthetic pigments. Using the ascorbate reduction method, the upper limit to the spin label half-life is tau 1/2 approximately 4 min at 0 degrees C. The flip-flop rate is rapid compared to that of a phospholipid spin label under the same conditions. The presence or absence of magnesium in the center of the porphyrin ring had no measurable effect on the flip-flop rate.     

Lipid Synthesis and Abundance of Acetyl CoA Carboxylase in Isochrysis galbana (Prymnesiophyceae) Following Nitrogen Starvation

Fatty acid content and the rate of lipid synthesis were measuredin the marine prymnesiophyte Isochrysis galbana grown undernitrogen starvation and in cultures recovering from nitrogendeprivation. Nitrogen starvation imposed a reduction in cellularsoluble protein content, variation in fatty acid compositionand reduction in the in vitro activity of the enzyme acetylCoA carboxylase. An increase in total fatty acid content isattributed to a differential reduction in cell division andthe rate of lipid synthesis. Recovery from nitrogen deprivationwas characterized by an increase in cellular soluble proteincontent and in the rate of lipid synthesis. Although the invitro activity of acetyl CoA carboxylase increased as the culturesrecovered from nitrogen starvation, the total cellular fattyacid content decreased, evidently due to an acceleration incell division. The relative cellular pool size of acetyl CoAcarboxylase determined by immunoblotting decreased under nitrogenstarvation conditions and increased as cells recovered fromit. Cellular accumulation of acetyl CoA carboxylase during recoveryfrom nitrogen starvation is ascribed to de novo synthesis ofthe enzyme that takes place in the cytoplasm. However, photosyntheticproteins such as ribulose bisphosphate carboxylase are synthesizedearlier than acetyl CoA carboxylase in the recovery process. (Received June 12, 1992; Accepted September 21, 1992)     

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.     

Motion of spin-labeled fatty acids in murine macrophages relation to cellular phagocytic activity

Macrophage membrane fluidity was investigated with respect to cellular phagocytic activity through the use of fatty acid spin labels.Spin-labeled fatty acid derivatives were incorporated into intact mouse peritoneal macrophages by exchange from bovine serum albumin. The electron spin resonance (ESR) spectra of the spin-labeled fatty acids in the macrophages showed a pronounced temperature dependence and a decrease in the hyperfine splittings ($\text{2T}{}_{\mathrm{|}}$) of the spectra as the nitroxide radical was moved away from the polar head group of the fatty acid derivatives.Spin-labeled macrophages underwent a time- and temperature-dependent decay, which was inhibited by preincubating the cells with mercuric chloride, heating at 56 °C, or by fixing them with 0.25 % glutaraldehyde.No correlation between the phagocytic activity of macrophages and membrane freedom of motion could be demonstrated. Treatment of macrophages with anti-macrophage serum or extended in vitro cultivation inhibited cellular phagocytic activity but exerted no effect on the motional freedom of the macrophage membrane. Enrichment of the fatty acid composition of the macrophage membrane with $\text{cis-}$ or $\text{trans-}\text{unsaturated}$ fatty acids had striking effects on cellular phagocytic activity, while no significant changes could be detected in the freedom of motion of incorporated fatty acid spin labels at the degree of specific enrichment achieved here. Thus no correlation between cellular phagocytic activity and lipid motion could be detected.     

Spin-label studies on the aqueous regions of phospholipid multilayers.

Water-soluble spin labels were used to study dimyristoyllecithin (DML) phospholipid multilayers. Previous studies report that there is a "bound" water region associated with dimyristoyllecithin containing about 10 molecules of water per phospholipid, a "trapped" water region located between the lamellae containing approximately 11 molecules per phospholipid, and a "ftion show that certain water-soluble spin-label mol-cules have their motional properties differentially modified by these three water environements. Furthermore, the labels also reveal the onset of lipid-phase transitions even though they have high water solubility. A phosphate-containing spin label demonstrated strong an isotropic motion in the lipid-water system above the phase transition but not below. The addition of cholesterol to the DML-water system removed the anisotropic motion of 2,2,6,6-tetramehtyl-4-phosphopiperidine-N-oxyl (Tempophosphate) and obscured the detection bound, trapped, and free water. In addition to the change-charge interactions between Tempophosphate and DML, two other spin labels were used both in the charged and uncharged states. 2,2,6,6-Tetramethyl-4-aminopiperidine-N-oxyl (Tempamine) in the charged state showed extremely strong anisotropic motion, presumably due to the interaction between the charged amine and the phosphate group of DML. When only partially charged, Tempamine showed much less anisotropic motion. PCA was analyzed at pH values where the carboxyl group was protonated and unprotonated. The resulting interaction was different at the two pH values. These water-soluble spin labels mimic ionic or nonionic solutes. Upon freezing, the spin labels are shown to be expelled from the ice regions into the remaining aqueous regions. The usefulness of this approach in studying solute behavior when freezing occurs and potential studies involving aqueous regions of cytoplasm are considered.     

Dynamic spin label study of the barstar-barnase complex

The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.