Transfer of stearic acids from albumin to polymer-grafted lipid containing membranes probed by spin-label electron spin resonance

Human serum albumin (HSA) has been spin-labelled with stearic acids having the nitroxide moiety attached to the hydrocarbon chain either at the 5th or at the 16th carbon atom (n-SASL, n = 5 and 16, respectively) with respect to the carboxyl groups. Its interaction with sterically stabilized liposomes (SSL) composed of dipalmitoylphosphatidylcholine (DPPC) mixed with submicellar content of poly(ethylene glycol:2000)-grafted dipalmitoyl phosphatidylethanolamine (PEG:2000-DPPE) has been studied by conventional electron spin resonance (ESR) spectroscopy. In the absence of bilayer membranes, the ESR spectra of nitroxide stearic acids non-covalently bound to HSA are single component powder patterns, indicative of spin labels undergoing temperature dependent anisotropic motion in the slow motional regime on the conventional ESR timescale. The adsorption of HSA to DPPC bilayers results in two component ESR spectra. Indeed, superimposed to an anisotropic protein-signal appears a more isotropic signal due to the labels in the lipid environment. This accounts for the transfer of fatty acids from the protein to DPPC bilayers. Two spectral components with different rotational mobility are also singled out in the spectra of n-SASL bound to HSA when DPPC/PEG:2000-DPPE mixtures are present in the dispersion medium. The fraction, f(L)(16-SASL), of spin labels transferred from the protein to lipid/polymer-lipid lamellar membranes has been quantified performing spectral subtraction. It is found that f(L)(16-SASL) decreases on increasing the content of the polymer-lipid mixed with DPPC. It is strongly reduced in the low-density mushroom regime and levels off in the high-density brush regime of the polymer-lipid content as a result of the steric stabilization exerted by the PEG-lipids. Moreover, the fraction of transferred fatty acids from HSA to SSL is dependent on the physical state of the lipid bilayers. It progressively increases with increasing the temperature from the gel to the liquid-crystalline lamellar phases of the mixed lipid/polymer-lipid membranes, although such a dependence is much weaker in the brush regime.     

Interaction between glycophorin and ganglioside GM1 on liposomal membranes. Effect of the interaction on the susceptibility of membranes to HVJ

Liposomes could bind and fuse efficiently to human erythrocytes in the presence of HVJ when they contained glycophorin isolated from human erythrocytes (Umeda, M., et al. (1983) J. Biochem. 94, 1955). In the present work we demonstrated that HVJ-induced fusion between liposomes containing glycophorin and erythrocytes was suppressed when GM1 coexisted with glycophorin in the same liposomal membranes. Asialo-GM1 and other gangliosides such as GM3 and sialosylparagloboside did not affect the fusion between the liposomes and erythrocytes. An intermolecular interaction between glycophorin and GM1 was suggested by the ESR spectrum obtained from liposomes containing glycophorin and a ganglioside GM1 analog carrying a nitroxyl spin label in the fatty acyl chains (5SL-gangliosidoide). The overall splitting value (2A parallel) observed in the ESR spectrum of liposomes containing 5SL-gangliosidoide increased with increase of the amount of glycophorin, whereas 2A parallel of spin-labeled phosphatidylcholine was not changed. The increase of 2A parallel of 5SL-gangliosidoide suggests that the mobility of the fatty acyl chain of the gangliosidoide was restricted by the interaction with glycophorin. It can be concluded that GM1 located near glycophorin, a receptor of the virus, interferes with the activity of viral F protein, inhibiting the fusion of liposome to erythrocyte.     

An electron spin resonance spin-label study of lipophilin in oriented phospholipid bilayers

Model membranes consisting of dimyristoyl phosphatidylcholine and a hydrophobic protein from bovine myelin, lipophilin, were studied using the cholesterol-resembling cholestane ESR spin label. Orientation of the membranes made it possible to deconvolute the spectra into two fractions, one of oriented spin labels reflecting phospholipid bilayer of high order, and one of isotropically tumbling spin labels ascribed to the lipid fraction surrounding the protein molecule (boundary lipid). This isotropic tumbling is different from the behavior of phospholipid molecules near the protein, which retain some degree of order, and indicates that the boundary lipid fraction in our model system forms a rather fluid environment for the protein. A nonlinear relation was found between protein concentration and amount of boundary spin labels. Addition of cholesterol decreases the amount of boundary spin labels. Both findings form evidence for a preferential binding of cholesterol by the membrane protein.     

Effect of fatty acyl chain length of phosphatidylcholine on their transfer from liposomes to erythrocytes and transverse diffusion in the membranes inferred by TEMPO-phosphatidylcholine spin probes

TEMPO-phosphatidylcholine (PC) spin probes which have homologous saturated acyl chains of 10, 12, 14 and 16 carbon atoms, were synthesized as analogues of PC. Transfer of TEMPO-PCs from liposomal membrane to the ghost membrane of human erythrocyte and transverse diffusion of TEMPO-PCs within the membrane of intact erythrocytes were determined by measurement of spontaneous increase and decrease in signal amplitude of an anisotropic triplet spectrum, due to dilution of the label by natural phospholipid of the membrane and reduction of the label by the cytoplasmic content of the erythrocyte, respectively. TEMPO-PC molecules in TEMPO-PC liposomes, except dipalmitoyl TEMPO-PC, were rapidly incorporated into the ghost membrane by incubation at 37 degrees C; the PC having shorter acyl chains was transferred faster. The cytoplasmic content of the erythrocyte rapidly reduced the nitroxide radical of the spin probe. The central peak height of ESR signal was once increased by incorporation of TEMPO-PC into the erythrocyte membrane and then was spontaneously decreased during further incubation at 37 degrees C. This decrease indicates that PC molecules traverse from the outer to the inner layer of the membrane lipid bilayer. The decrease of signal amplitude was faster with PC of shorter acyl chain. These findings suggest that both transfer between membranes and transverse diffusion in the membrane may be favored to the PC species with shorter acyl chains.     

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.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using 2H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and 2H-NMR order parameters in membranes.     

Analysis of existing models of spin label movement during spin labeling of biological macromolecules

The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes. A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This 'stiffening' effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35 degrees C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This correspond to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10 degrees C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes: A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This ‘stiffening’ effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35°C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Transfer of cholestane spin label between lipid bilayer membranes and its molecular motion in membranes.

The relation between the molecular motion of a steroid in lipid membranes and the transfer rate between membranes was examined using radioactive cholestane spin label. Order parameters of the molecule were determined in bilayers composedof dipalmitoylglycerophosphocholine or egg yolk phosphatidylcholine at various temperatures. The line widths of the ESR signal of the cholestane spin label in membranes, which depend upon the rate of molecular axial rotation in the membranes, were also measured. The temperature dependences of these two parameters and of the transfer rate suggest a close correlation between the rate of molecular axial rotation and the transfer rate.     

The conformations of nitroxide-labelled fatty acid probes of membrane structure as studied by 2H-NMR

The electron spin resonance (ESR) spectrum of a nitroxide spin probe intercalated in a membrane is influenced by the amplitude of anisotropic motion of the nitroxide group and by the geometry of the oxazolidine ring of the nitroxide. In the analysis of the ESR spectra of nitroxide-labelled fatty acid probes, it is generally assumed that the five-membered oxazolidine ring system is oriented rigidly perpendicular to the long molecular axis of the probe. This assumption is tested in the present study, using ²H-NMR of specifically deuterium-labelled nitroxide spin probes. Evidence is presented that the nitroxide does not display the assumed geometry in membranes. The departure from this geometry depends on the position of the nitroxide label on the acyl chain, with a more pronounced departure for position 5 relative to position 12. These and previous data provide an explanation for the discrepancies between spin-probe ESR and ²H-NMR order parameters in membranes.     

ESR measurement of rapid penetration of DMPO and DEPMPO spin traps through lipid bilayer membranes

The passive permeation rates of DMPO and DEPMPO spin traps and their hydroxyl radical adducts through liposomal membranes were measured using ESR spectroscopy. For the spin traps, we measured the time-dependent change in the signal intensity of the OH-adduct, which is formed by a reaction between the penetrated spin trap and hydroxyl radicals produced by the UV-radiolysis of H(2)O(2) inside the liposomes. The hydroxyl radicals produced outside the liposomes were quenched with polyethylene glycol. For the OH-adduct, pre-formed adduct was mixed with liposomes and the time-dependent change of the ESR signal was measured in the presence of a line-broadening reagent outside the liposomes to make the signal outside the liposomes invisible. Both the spin traps and their OH-adducts diffused across the lipid membranes rapidly and reached equilibrium within tens of seconds. These findings suggest that if used for the detection of free radicals inside cells, these spin traps should be well distributed in cells and even in organelles.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Dynamic properties of the haptenic site of lipid haptens in phosphatidylcholine membranes. Their relation to the phase transition of the host lattice.

The relation between the dynamic properties of the haptenic site of lipid haptens and the phase transition of the host lattice was investigated using head group spin-labeled phosphatidylethanolamines, that is, spin-label lipid haptens (Br?let, P., and H. M. McConnell, 1976, Proc. Natl. Acad. Sci. USA., 73:2977-2981; Br?let, P., and H. M. McConnell, 1977, Biochemistry, 16:1209-1217). The electron spin resonance (ESR) spectra of the lipid haptens in liposomal membranes showed three narrow resonance lines, whose widths and hyperfine splitting values suggested that the haptenic site, i.e., the spin-label moiety, should be exposed in the water phase. The line width of each peak depended on the host lipid species and on the incubation temperature. A temperature study using dipalmitoylphosphatidylcholine (DPPC) liposomes showed that the dynamic properties of the haptenic site were related to the main phase transition and the subphase transition of the host lattice but not to the prephase transition. The angular amplitudes of the tumbling motion of the haptenic site were estimated using oriented multibilayer systems. The angular amplitude of dipalmitoyl-phosphatidyl-N-[[N-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidinyl)-carbamoyl]-methyl]-ethanolamine in DPPC membranes was 63 degrees at 2 degrees C, and it increased slightly with an increase in temperature regardless of the phase transition of the host lattice. The value for egg phosphatidylcholine (PC) at 25 degrees C was the same as for DPPC above its main phase transition temperature. Rotational correlation time analysis showed that the axial rotation of the haptenic site was preferable to the tumbling motion of the rotational axis, and the predominance depended on the phase transition, Lc----L beta' and P beta'----L alpha. Elongation of the spacer arm between the haptenic site and phosphate increased the angular amplitude of the tumbling motion but reduced the effect of the host lattice. Spin-label lipid haptens with unsaturated fatty acyl chains were distributed heterogeneously in DPPC membranes, whereas those with the same fatty acyl chain as the host lattice were distributed randomly. The ESR spectrum of a lipid hapten under its prephase transition temperature showed two components, broad and narrow. This suggests that at least two different domains, a hapten-rich domain and a hapten-poor one, may coexist in membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

Incorporation of a ganglioside and spin-labeled ganglioside analogue into cell and liposomal membranes

When an aqueous solution of a spin-labeled "two tail" gangliosidoid was incubated with liposomes or sheep erythrocytes, the broad single resonance line in the ESR spectrum disappeared and a signal showing an anisotropic motion appeared, indicating that the spin-labeled "two tail" gangliosidoid in the micellar state was transferred to the lipid phase of the acceptor membranes. The transfer was temperature- and time-dependent, irrespective of the acceptor membranes, indicating that the rate of transfer is determined by the escape of monomers from the micelles. The kinetics and temperature-dependence of the association of ganglioside II3NeuAc-GgOse4Cer with sheep erythrocytes was very similar to that of the "two tail" gangliosidoid, indicating that parts of ganglioside II3NeuAc-GgOse4Cer could be incorporated into the lipid phase of membranes via a similar mechanism.     

Interaction of small molecules with phospholipid bilayer membranes: A spin label study

Multilamellar spin labelled liposomes were prepared from dipalmitoyl or dimyristoyl phosphatidylcholine, dicetyl phosphate, and the spin probe 12-doxyl stearate methyl ester. The effects of a series of benzene and adamantane derivatives, on fatty acyl chain motion was measured through changes in the electron spin resonance spectra of these liposomes. All the compounds tested, increased lipid chain motion to a variable degree. In general, molecules possessing a polar group were more potent than those lacking such a group and lipophilicity per se correlated poorly with the relative order of these compounds. Within the adamantane series separating the polar group from the cage structure by the insertion of methylene groups further enhanced the capacity of the molecule to increase hydrocarbon chain mobility. These observations are consistent with the hypothesis that the location of the additive within the bilayer is the main determinant of its effectiveness in increasing fatty acyl chain motion.     

Electron spin resonance analysis of irreversible changes induced by calcium perturbation of erythrocyte membranes.

Introduction of calcium during hemolysis of erythrocytes causes irreversible membrane changes, including protein aggregation. These changes have been investigated by incorporation of one protein and three fatty acid spin label probes into washed membranes from erythrocytes hemolyzed with a range of Ca2+ concentrations. Electron spin resonance spectra of the lipid probes were analyzed for changes in the order parameters, isotropic coupling constants and mean angular deviations of the lipid hydrocarbon chains. The results generally indicated an increased freedom of mobility of the probes with increased Ca2+ concentration during hemolysis, but the response of each probe showed a different concentration dependence. The maximal response was obtained with the I(5, 10) probe. Variations in the responses were interpreted to reflect different modes of protein-lipid or protein-probe interactions arising from Ca2+ -induced membrane protein alterations. Spectra from membranes treated with the protein spin label showed an increased ratio of immobilized to mobile label with increased Ca2+ concentrations at hemolysis. This is consistent with the membrane protein aggregation phenomena previously observed. It is suggested that the increased protein-protein interactions formed as a result of calcium treatment permit an increased lipid mobility in the membrane regions monitored by the fatty acid probes.