Stearic Acid Spin Labels Research Articles

Depth dependence of the perturbing effect of placing a bulky group (oxazolidine ring spin labels) in the membrane on the membrane phase transition

Electron paramagnetic resonance (EPR) and differential scanning calorimetry (I)SO have been used to study the effect on the phase transition of dimyristoylphosphatidylcholine membranes of incorporating various stearic acid spin labels (SASL's) that contain the bulky oxazolidine ring at various positions along the stearyl chain. SASL's lowered the phase transition temperature and decreased the size of the cooperative unit, with the effects stronger in the order of 9-> 12-> 5-> 16-SASL > stearic acid (no label). Incorporation of stearic acid without the spin label slightly increases the phase transition temperature. Incorporation of 9-SASL (3 mol% of lipid) decreased the transition temperature by 1.8C and the cooperative unit to 115 of that without the spin label, while the effect of 16-SASL was slight. The effect on transition enthalpy was small. It is concluded that the perturbing effect of placing a bulky group on the alkyl chain on phase transition is through inducing packing defects in the gel-phase.

Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes.

We have used spin-label EPR spectroscopy to examine possible alterations in protein-protein interactions that accompany the activation of the cardiac sarcoplasmic reticulum (SR) Ca-ATPase following the phosphorylation of phospholamban (PLB). Using a radioactive derivative of a maleimide spin label (MSL), we have developed conditions for the selective spin-labeling of the Ca-ATPase in both native cardiac and skeletal sarcoplasmic reticulum membranes. The rotational dynamics of the cardiac and skeletal Ca-ATPase isoforms in native SR membranes were measured using saturation transfer EPR. We report that the phosphorylation of PLB in cardiac SR results in a (1.8 +/- 0.2)-fold reduction in the overall rotational mobility of the Ca-ATPase. The alteration in the rotational dynamics of the Ca-ATPase is the direct result of the phosphorylation of PLB, and is not related to the phosphorylation of the Ca-ATPase or any other SR proteins since no alteration in the ST-EPR spectrum is observed as a result of conditions that phosphorylate the cardiac Ca-ATPase with ATP. Neither do the use of conditions that activate the Ca-ATPase in cardiac SR result in the alteration of the rotational dynamics or catalytic properties of the Ca-ATPase in skeletal SR where PLB is not expressed. Measurements of the rotational dynamics of stearic acid spin labels (SASL) incorporated into cardiac SR membranes with a nitroxide at the 5- and 12-positions using conventional EPR indicate that there is virtually no difference in the lipid acyl chain dynamics in cardiac SR membranes upon the phosphorylation of PLB. These results indicate that the decrease in the rotational dynamics of the Ca-ATPase in cardiac SR membranes associated with the phosphorylation of PLB is related to enhanced interactions between individual Ca-ATPase polypeptide chains due to (i) an alteration in the spatial arrangement of cardiac Ca-ATPase polypeptide chains within a defined oligomeric state or (ii) increased protein-protein associations. We suggest that altered interactions between Ca-ATPase polypeptide chains and PLB serves to modulate the activation barrier associated with calcium activation of the Ca-ATPase in cardiac SR membranes.

Adaptive changes in lipid composition of skeletal sarcoplasmic reticulum membranes associated with aging

We have undertaken a detailed examination of changes associated with aging in lipid composition and corresponding physical properties of hindlimb skeletal sarcoplasmic reticulum (SR) membranes isolated from young (5 months), middle-aged (16 months), and old (28 months) Fischer strain 344 rats. Silica gel HPLC chromatography was used to separate phospholipid headgroup species. Subsequent reversed-phase HPLC was used to resolve fatty acid chain compositions of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species. For all three phospholipid pools, significant age-related variations are observed in the abundance of multiple molecular species, particularly those having polyunsaturated fatty acid chains. Using mass spectrometry (fast atom bombardment and tandem techniques) to distinguish ester- from ether-linked phosphatidylethanolamine species, we demonstrate that overall plasmenylethanolamine content is substantially increased with age, from 48 mol% to 62 mol%. A substantial increase is also observed in the single molecular species 18:0–20:4 phosphatidylinositol suggesting implications for signalling pathways. In addition, associated with senescence we find a significant increase in the rigidifying lipid, cholesterol. Despite these changes in lipid composition of different aged animals, the average bilayer fluidity examined at several bilayer depths with stearic acid spin labels, is not altered. Neither do we find differences in the rotational mobility of maleimide spin-labeled Ca 2+-ATPase, as determined from saturation-transfer electron paramagnetic resonance, which is sensitive to both the fluidity of lipids directly associated with the Ca 2+-ATPase and to its association with proteins.

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.

Spin label EPR study of lipid solvation of supramolecular photosynthetic protein complexes in thylakoids

Lipid-protein association in the chloroplast membrane and its various thylakoid fractions from higher plants, namely pea and maize, rich in Photosystem I (PSI) and Photosystem II (PSII), respectively, were studied using EPR spectroscopy of spin-labelled lipid molecules. All the EPR spectra consisted of two spectral components corresponding to bulk fluid lipids and solvation lipids motionally restricted at the hydrophobic surface of membrane proteins. Spin-labelled stearic acid and phosphatidylglycerol exhibited marked selectivity towards the supramolecular protein complexes of both PSI and PSII although to different extent. In addition, lipid-protein titration experiments are described for partially delipidated PSII-enriched membrane fractions of pea chloroplasts, incorporating unlabelled egg phosphatidylcholine prior to or after the incorporation of spin-labelled lipids. Two sets of solvation sites were resolved by timed labelling experiments and a significant result of these studies was that a well-defined population of solvation sites (approx. 100 mol lipids/820 kDa protein) was rapidly exchanged by laterally diffusing membrane lipids, while other solvation sites (approx. 50 mol lipids/820 kDa protein) were exchanged much slower or not exchanged at all.

Partitioning of Stearic Acid Spin Labels into Phospholipid Bilayers with Different Fluidities

The difference of spin label density between the phases with different fluidities was studied in multilamellar DPPC liposomes. ESR spectra of stearic acid spin labels of unprotonated form were observed. The spectral intensity due to the labels in lipid was measured on comparing to that due to the labels in the buffer solution. The intensity ratio of these two lines was calculated in both the gel and liquid crystalline phases. The label density of 16-SAL in the liquid crystalline phase is 2.8 times more concentrated than that in the gel phase. The values are 1.85 for 5-SAL and 5.6 for 12-SAL. The application of these values to the case when two regions with different fluidities coexist in a lipid bilayer was studied.

Effects of probucol on phase transition and fluidity of phosphatidylcholine membranes: A spin label study

Spin labeling methods were applied to study the structure and dynamics of phosphatidylcholine membranes as a function of temperature and the mole fraction of probucol. Multilamellar liposomes made of dimyristoylphosphatidyclcholine, dipalmitoylphosphatidylcholine both saturated, and egg yolk phosphatidylcholine, an unsaturated membrane, were used. In fluid phase membranes probucol was found to increase the order and decrease the motional freedom of alkyl chains of lipids as shown with stearic acid spin labels. The effect of probucol on order and motional freedom is more pronounced in the membrane center (16-doxylstearic acid spin label position) than in the near polar headgroup region (5-doxylstearic acid spin label position). The presence of unsaturation in alkyl chains significantly decreased the ordering effect of probucol. The main phase transition temperature of saturated bilayers was lowered by 2°C in the presence of 3 mol% of probucol and significantly broadened at higher concentrations as measured with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) partitioning. Also, pretransition was no longer observed in the presence of probucol. In gel phase membranes, the effect of probucol was complex. Close to the main phase transition the motion of alkyl chains was increased, showing a regulatory effect of probucol on membrane fluidity. It is proposed that probucol is located in the membrane center as opposed to vitamin E, which locates its phenolic -OH group at the membrane surface; therefore, it inhibits lipid peroxidation in this region which is less accessible to vitamin E.

Hydrophobic Barriers of Lipid Bilayer Membranes Formed by Reduction of Water Penetration by Alkyl Chain Unsaturation and Cholesterol

The hydrophobicity profiles across phosphatidylcholine (PC)-cholesterol bilayer membranes were estimated in both frozen liposome suspensions and fluid-phase membranes as a function of alkyl chain length, unsaturation, and cholesterol mole fraction. A series of stearic acid spin labels, with the probe attached to various positions along the alkyl chain, cholesterol-type spin labels (cholestane and androstane spin labels), and Tempo-PC were used to examine depth-dependent changes in local hydrophobicity, which is determined by the extent of water penetration into the membrane. Local hydrophobicity was monitored primarily by observing the z component of the hyperfine interaction tensor (Az) of the nitroxide spin probe in a frozen suspension of the membrane at -150 degrees C and was further confirmed in the fluid phase by observing the rate of collision of Fe(CN)6(3-) with the spin probe in the membrane using saturation recovery ESR. Saturated-PC membranes show low hydrophobicity (high polarity) across the membrane, comparable to 2-propanol and 1-octanol, even at the membrane center where hydrophobicity is highest. Longer alkyl chains only make the central hydrophobic regions wider without increasing the level of hydrophobicity. Introduction of a double bond at C9-C10 decreases the level of water penetration at all locations in the membrane, and this effect is considerably greater than the cis configuration than with the trans configuration. Incorporation of cholesterol (30 mol %) dramatically changes the profiles; it decreases hydrophobicity (increases water penetration) from the polar headgroup region to a depth of approximately C7 and C9 for saturated- and unsaturated-PC membranes, respectively, which is about where the bulky rigid steroid ring structure of cholesterol reaches in the membrane. Membrane hydrophobicity sharply increases at these positions from the level of methanol to the level of pure hexane, and hydrophobicity is constant in the inner region of the membrane. Thus, formation of effective hydrophobic barriers to permeation of small polar molecules requires alkyl chain unsaturation and/or cholesterol. The thickness of this rectangular hydrophobic barrier is less than 50% of the thickness of the hydrocarbon regions. Results obtained in dioleoyl-PC-cholesterol membranes in the fluid phase are similar to those obtained in frozen membranes. These results correlate well with permeability data for water and amino acids in the literature.

Quenching of merocyanine 540 triplet state by nitroxyl radicals in liposomal systems: a laser flash photolysis study

Laser flash photolysis experiments were undertaken to investigate the interaction between stearic acid nitroxide spin probes and photoexcited merocyanine 540 (MC540) in dimyristoyl- l-α-phosphatidylcholine liposomes (membrane model). The measurements of the paramagnetic signal decay kinetics of four different spin-labelled stearic acids ( n-DSA) show that the direct interaction between the dye and the probe is affected by the position of the nitroxyl group along the carbon chain. Laser flash photolysis results reveal a significant decrease in the MC540 triplet lifetime in the presence of n-DSA, the effect depending on the depth at which the nitroxyl moiety is localized in the bilayer. Previous results on the rate of disappearance of the electron spin resonance (ESR) nitroxide signal on continuous photolysis of the same systems do not show the same dependence on the localization of the nitroxyl moiety in the liposome. Although the MC540 triplet state seems to be implicated in the reaction process, the results suggest that ESR and laser flash experiments demonstrate two different kinds of mechanism.

Protein and lipid rotational dynamics in cardiac and skeletal sarcoplasmic reticulum detected by EPR and phosphorescence anisotropy.

We have used time-resolved phosphorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy to detect the rotational dynamics of the Ca-ATPase and its associated lipids in dog cardiac sarcoplasmic reticulum (DCSR), in comparison with rabbit skeletal SR (RSSR), in order to obtain insight into the physical bases for different activities and regulation in the two systems. Protein rotational motions were studied with time-resolved phosphorescence anisotropy (TPA) of erythrosin isothiocyanate (ERITC) and saturation-transfer EPR (ST-EPR) of a maleimide spin-label (MSL). Both labels were attached selectively and rigidly to the Ca-ATPase. Lipid rotational motions were studied with conventional EPR of stearic acid spin-labels. As in previous studies on RSSR, the phosphorescence anisotropy decays of both preparations at 4 degrees C were multiexponential, due to the presence of different oligomeric species. The rotational correlation times for the different rotating species were similar for the two preparations, but the total decay amplitude was substantially less for cardiac SR, indicating that more of the Ca-ATPase molecules are in large aggregates in DCSR. ST-EPR spectra confirmed that the Ca-ATPase is less rotationally mobile in DCSR than in RSSR. Lipid probe mobility and fatty acid composition were very similar in the two preparations, indicating that the large differences observed in protein mobility are not due to differences in lipid fluidity. We conclude that the higher restriction in protein mobility observed by both ST-EPR and TPA is due to more extensive protein-protein interactions in DCSR than in RSSR.

Exchange rates at the lipid-protein interface of the myelin proteolipid protein determined by saturation transfer electron spin resonance and continuous wave saturation studies

The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.

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.

Structural and functional modifications induced by ketamine on synaptosomes in the rat

This study correlates previous observations on the effects of a general anaesthetic (2-chlorophenyl-2-methylaminocyclohexanone, trade name Ketalar), administered in vivo on the functional properties of synaptosomes from the brain of the rat, with structural modifications detected using freeze-fracture electron microscopy. The anaesthetized rats exhibited a fluidization of the synaptic membranes, as probed by stearic acid spin labels, accompanied by a decrease in the activity of acetylcholinesterase. The freeze-fracture images of synaptosomes from anaesthetized rats showed a clear statistical increase in the number of vesicles with aggregated intramembrane particles, without any significant change of the particle diameter. The hypothesis that a perturbation of the lipid protein interaction is a primary effect, in the mechanism of action of anaesthetics, is supported at present only by indirect evidence.

Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance.

We have performed electron paramagnetic resonance (EPR) experiments on nitroxide spin labels incorporated into rabbit skeletal sarcoplasmic reticulum (SR), in order to investigate the physical and functional interactions between melittin, a small basic membrane-binding peptide, and the Ca-ATPase of SR. Melittin binding to SR substantially inhibits Ca(2+)-dependent ATPase activity at 25 degrees C, with half-maximal inhibition at 9 mol of melittin bound per mole of Ca-ATPase. Saturation transfer EPR (ST-EPR) of maleimide spin-labeled Ca-ATPase showed that melittin decreases the submillisecond rotational mobility of the enzyme, with a 4-fold increase in the effective rotational correlation time (tau r) at a melittin/Ca-ATPase mole ratio of 10:1. This decreased rotational motion is consistent with melittin-induced aggregation of the Ca-ATPase. Conventional EPR was used to measure the submicrosecond rotational dynamics of spin-labeled stearic acid probes incorporated into SR. Melittin binding to SR at a melittin/Ca-ATPase mole ratio of 10:1 decreases lipid hydrocarbon chain mobility (fluidity) 25% near the surface of the membrane, but only 5% near the center of the bilayer. This gradient effect of melittin on SR fluidity suggests that melittin interacts primarily with the membrane surface. For all of these melittin effects (on enzymatic activity, protein mobility, and fluidity), increasing the ionic strength lessened the effect of melittin but did not alleviate it entirely. This is consistent with a melittin-SR interaction characterized by both hydrophobic and electrostatic forces. Since the effect of melittin on lipid fluidity alone is too small to account for the large inhibition of Ca-ATPase rotational mobility and enzymatic activity, we propose that melittin inhibits the ATPase primarily through its capacity to aggregate the enzyme, consistent with previous observations of decreased Ca-ATPase activity under conditions that decrease protein rotational mobility.

EPR study of the hydrophobic interaction of spectrin with fatty acids

The hydrophobic interaction between spin-labelled stearic acid and spectrin was studied by electron paramagnetic resonance (EPR) and fluorescence quenching. The results are quantitatively interpreted in terms of two types of binding site on spectrin. A comparison between the results of the EPR and fluorescence experiments show the drawback of the fluorescence method in bindin studies.

Resolution of phospholipid conformational heterogeneity in model membranes by spin-label EPR and frequency-domain fluorescence spectroscopy

We have utilized both fluorescent and nitroxide derivatives of stearic acid as probes of membrane structural heterogeneity in phospholipid vesicles under physiological conditions, as well as conditions of varying ionic strengths and temperatures where spectral heterogeneity has been previously observed and attributed to multiple ionization states of the probes. To identify the source of this spectral heterogeneity, we have utilized complimentary measurements of the relaxation properties (lifetimes) and motion of both (a) spin labeled and anthroyloxy derivatives of stearic acid (i.e., SASL and AS) and (b) a diphenylhexatriene derivative of phosphatidylcholine (DPH-PC) in single component membranes containing dimyristoylphosphatidylcholine (DMPC). We use an 15N stearic-acid spin label for optimal sensitivity to membrane heterogeneity. The lifetime and dynamics of the fluorescent phospholipid analogue DPH-PC (with no ionizable groups over this pH range) were compared with those of AS, allowing us to discriminate between changes in membrane structure and the ionization of the label. The quantum yield and rotational dynamics of DPH-PC are independent of pH, indicating that changes in pH do not affect the conformation of the host phospholipids. However, both EPR spectra of SASL and the lifetime or dynamics of AS are affected profoundly by changes in solution pH. The apparent pKa's of these two probes in DMPC membranes were determined to be near pH 6.3, implying that at physiological pH and ionic strength these stearic-acid labels exist predominantly as a single ionized population in membranes. Therefore, the observed temperature- and ionic-strength-dependent alterations in the spectra of SASL as well as the lifetime or dynamics of AS in DMPC membranes at neutral pH are due to changes in membrane structure rather than the ionization of the probes. The possibility that ionic gradients across biological membranes induce alterations in phospholipid structures, thereby modulating lipid-protein interactions is discussed.

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 distrophy (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.

Mapping of collision frequencies for stearic acid spin labels by saturation-recovery electron paramagnetic resonance

Short pulse saturation-recovery electron paramagnetic resonance methods have been used to measure interactions of 14N:15N stearic acid spin label pairs in multilamellar liposomal dispersions composed of dimyristoyl-phosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC). Pairs consisting of various combinations of [14N]-16-, [14N]-12- or [14N]-5-doxylstearate, and [15N]-16-, [15N]-12-, or [15N]-5-doxylstearate were studied. SR experiments were performed at 27 degrees and 37 degrees C, and recovery signals were analyzed for initial conditions and multiexponential time constants by computer fitting using a damped least-squares approach. The time constants contain combinations of the electron spin lattice relaxation time, Tle, for each member of the spin-label pair, and the Heisenberg exchange rate constant, Kx. Spin-lattice relaxation times for each of the 14N and 15N stearic acid spin labels were determined, and it is noted that Tle for a given 15N-SASL was always slightly greater than that of the corresponding 14N-SASL. From Kx the bimolecular collision frequency was calculated, providing a detailed picture of molecular interactions. For both lipid systems the bimolecular collision rates were ordered as 12:5 less than 16:5 less than 5:5 less than 16:12 less than 12:12 less than 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in DEPC. For the 16:16, 12:12, and 16:12 pairs, Kx was approximately 30% greater in DMPC than in DEPC, a significantly greater difference than is observed by conventional EPR methods. Further confirmation of the existence of vertical fluctuation of nitroxide moieties that are at the 16- (or 12) position toward the polar surfaces was obtained, with the frequency of 16:5 (or 12:5) interactions ~40% of the 16:16 (or 12:12) interaction frequency. In both DMPC and DEPC, Kx for homogeneous pairs increases as the doxyl moiety is moved further down the alkyl chain (i.e.,5:5 < 12:12 < 16:16), suggesting that steric effects or the local rotational mobility of the nitroxide group influence the frequency at which spin exchange occurs.

Fluidity of the phospholipid bilayer of the endometrium at the time of implantation of the blastocyst — A spin label study

The endometrial phospholipid bilayer is shown to be in a highly fluid and polar state at the time of implantation in Mus musculus as evidenced from a spin label study using the stearic acid spin label, 5-doxyl stearate. The positive correlation observed between the superoxide anion radical levels and the fluidity and polarity state of the endometrial cells points towards the possibility of the mediation of this condition of unsaturation of fatty acids to be an act of the superoxide anion radical.

A study of the effect of general anesthetics on lipid-protein interactions in acetylcholine receptor-enriched membranes from Torpedo nobiliana [as measured by] using nitroxide spin-labels

Stearic acid, phosphatidylcholine, and phosphatidylglycerol nitroxide spin-labels were used to probe the effect of 1-hexanol, urethane, diethyl ether, and ethanol on lipid-protein interactions in nicotinic acetylcholine receptor (nAcChoR) rich membranes from Torpedo nobiliana. For stearic acid spin-labeled at the C-14 position of the sn-1 acyl chain, 1-hexanol induced little change (over a wide concentration range, 0-16.7 mM) in either the ESR line shape or the proportion of motionally restricted spectral component from labels probing the protein interface. The main effect of 1-hexanol was limited to an increase in the mobility of stearic acid spin-labels probing the non-protein-associated environment. In contrast, for C-14 phosphatidylcholine spin-label, 1-hexanol decreased the fraction of spin-labels motionally restricted at the protein interface from 0.33 without 1-hexanol to 0.20 with 16.7 mM 1-hexanol, with no change in the line shape of the spectral component of these labels. The ESR spectral line shape of the fluid component due to phosphatidylcholine labels in sites away from the protein interface displayed a gradual decrease in spectral anisotropy on addition of increasing amounts of 1-hexanol. At a concentration of 1-hexanol that desensitizes half the receptors, the fraction of motionally restricted phosphatidylcholine spin-label is reduced by approximately 15%. The effect of 1-hexanol on phosphatidylglycerol spin-labels was intermediate between these two cases. Similar effects were measured with other general anesthetics, including urethane, diethyl ether, and ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)