Thermotropic lipid phase separations in human erythrocyte ghosts and cholesterol-enriched rat liver plasma membranes

Summary Electron spin resonance (ESR) studies of human erythrocyte ghosts labeled with 5-nitroxide stearate, I(12,3), indicate that a temperature-dependent lipid phase separation occurs with a high onset at 38°C. Cooling below 38°C induces I(12,3) clustering. Similar phase separations were previously identified in human platelet and cholesterol-loaded [cholesterol/phospholipid molar ratio (C/P)=0.85] rat liver plasma membranes [L.M. Gordon et al., 1983;J. Membrane Biol. 76; 139–149]; these were attributed to redistribution of endogenous lipid components such that I(12,3) is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains. Further enrichment of rat liver plasma membranes to C/P ratios of 0.94–0.98 creates an artificial system equivalent to human erythrocyte ghosts (C/P=0.90), using such criteria as probe flexibility, temperature dependent I(12,3) clustering; and polarity of the probe environment. Consequently, cholesterol-rich and-poor domains probably exist in both erythrocyte ghosts and high cholesterol liver membranes at physiologic temperatures. The temperature dependence of cold-induced hypertonic lysis of intact human erythrocytes was examined by incubating cells in 0.9m sucrose for 10 min at 1°C intervals between 9 and 46°C (Stage 1), and then subjecting them to 0°C for 10 min (Stage 2). Plots of released hemoglobin are approx. sigmoidal, with no lysis below 18°C and maximal lysis above 40°C. The protective effect of low temperatures during Stage 1 may be due to the formation of cholesterol-rich domains that alter the bilayer distribution and/or conformation of critical membrane-associated proteins.     

Spin-label studies on rat liver and heart plasma membranes: do probe-probe interactions interfere with the measurement of membrane properties?

The structures of purified rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(12,3). ESR spectra were recorded with a 50 gauss field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The hyperfine splittings measured from the "expanded" spectra were significantly more precise than those obtained from the "unexpanded" spectra. Both procedures were used to study the effects of various I(12,3) probe concentrations on the spectra of liver and heart membranes, as well as the effects of temperature and CaCl2 additions on the spectra of liver membranes, and revealed the following: The polarity-corrected order parameters of liver (31 degrees) and heart (22 degrees) membranes were found to be independent of the probe concentration, if experimentally-determined low I(12,3)/lipid ratios were employed. The absence of obvious radical-interaction broadening in the unexpanded spectra indicated that "intrinsic" membrane properties may be measured at these low probe/lipid ratios. Here, "intrinsic" properties are defined as those which are measured when probe-probe interactions are negligible, and do not refer to membrane behavior in the absence of a perturbing spin label. At higher I(12,3)/lipid ratios, the order parameters of liver and heart membranes were found to substantially decrease with increasing probe concentration. The increase in the "apparent" fluidity of both membrane systems is attributed to enhanced radical interactions; however, an examination of these spectra (without reference to "low" probe concentration spectra) might incorrectly suggest that radical interactions were absent. For the membrane concentrations employed in these studies, the presence of "liquid-lines" (or "fluid components") in the unexpanded ESR spectra was a convenient marker of high probe concentrations. A thermotropic phase separation was observed in liver membranes between 19 degrees and 28 degrees. Addition of CaCl2 to liver plasma membrane [labelled with "low" I(12,3) concentrations] increased the rigidity of the membrane at 31 degrees and 37 degrees, without inducing a segregation of the probe in the bilayer. Previously reported data are discussed in relation to these results, and suggested minimal criteria for performing membrane spin label studies are included.     

Thermotropic lipid phase separation in the human immunodeficiency virus

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.     

Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase.

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions.     

Perturbations of liver plasma membranes induced by Ca2+ are detected using a fatty acid spin label and adenylate cyclase as membrane probes

Ca2+ decreased the lipid fluidity of rat liver plasma membranes labeled with 5-nitroxide stearate, I(12,3), as indicated by the order parameter (S). These effects form a reversible, saturable process with an association constant of 1 x 10(3) M-1. Arrhenius-type plots of S indicated that the lipid phase separation, present in the external leaflet of native membranes between 28 and 19 degrees C, is perturbed by mM Ca2+ such that the high temperature onset is elevated to 32-34 degrees C. Fluoride-stimulated adenylate cyclase was similarly inhibited by Ca2+ (ID50 = 1 mM) for the enzyme in membrane-bound or solubilized states. The glucagon-stimulated activity was more sensitive to Ca2+ inhibition with an ID50 of 0.2 mM. These inhibitory effects are due neither to perturbations of glucagon binding to its receptor nor to fluidity changes, but are instead attributed to direct Ca2+-enzyme interactions. Such binding desensitizes the enzyme to fluidity alterations induced by temperature elevation or benzyl alcohol addition. With Ca2+, Arrhenius plots of glucagon-stimulated activity indicated breaks at 32 and 16 degrees C, whereas those of fluoride-stimulated activity showed one break at 17 degrees C. Without Ca2+, Arrhenius plots exhibited one break at 28 degrees C for glucagon-stimulated activity, whereas fluoride-stimulated plots were linear. We propose that Ca2+ achieves these effects through asymmetric perturbations of the membrane lipid structure.     

Quinidine and melittin both decrease the fluidity of liver plasma membranes and both inhibit hormone-stimulated adenylate cyclase activity

Increasing concentrations of either quinidine or melittin gave a dose-dependent inhibition of both the glucagon- and fluoride-stimulated activities of adenylate cyclase in the liver plasma membranes. At similar concentrations these agents increased the order of liver plasma membranes as detected by a fatty acid ESR probe, doxyl stearic acid. This increase in bilayer order (decrease in 'fluidity') is suggested to explain the inhibitory action of quinidine on adenylate cyclase activity but only in part contributes to the inhibitory action of melittin on adenylate cyclase. Arrhenius plots of fluoride-stimulated activity became non-linear in the presence of either quinidine or melittin, with a single well-defined break occurring at around 12 degrees C in each instance. Arrhenius plots of the glucagon-stimulated activity also exhibited such a novel break at around 12 degrees C when either quinidine or melittin were present as well as exhibiting a break at around 28 degrees C, as was seen in the absence of these ligands. The fatty acid spin probe inserted into liver plasma membranes detected a novel lipid phase separation occurring at around 12 degrees C when either quinidine or melittin was present and showed that the lipid phase separation occurring at around 28 degrees C in native membranes was apparently unaffected by these ligands.     

Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.

1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only.     

Lipid-protein interactions in rat renal subcellular membranes: a biophysical and biochemical study

The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.     

Spin label studies on rat liver and heart plasma membranes: Effects of temperature,calcium, and lanthanum on membrane fluidity

The structures of rat liver and heart plasma membranes were studied with the 5-nitroxide stearic acid spin probe, I(1 2,3). The polarity-corrected order parameters (S) of liver and heart plasma membranes were independent of probe concentration only if experimentally determined low I(1 2,3)/lipid ratios were employed. At higher probe/lipid ratios, the order parameters of both membrane systems decreased with increasing probe concentration, and these effects were attributed to enhanced nitroxide radical interactions. Examination of the temperature dependence of approximate and polarity-corrected order parameters indicated that lipid phase separations occur in liver (between 19° and 28°C) and heart (between 21° and 32°C) plasma membranes. The possibility that a wide variety of membrane-associated functions may be influenced by these thermotropic phase separations is considered. Addition of 3.9 mM CaCl₂ to I(1 2,3)-labeled liver plasma membrane decreased the fluidity as indicated by a 5% increase in S at 37°C. Similarly, titrating I(1 2,3)-labeled heart plasma membranes with either CaCl₂ or LaCl₃ decreased the lipid fluidity at 37°C, although the magnitude of the La³⁺ effect was larger and occurred at lower concentrations than that induced by Ca²⁺; addition of 0.2 mM La³⁺ or 3.2 mM Ca²⁺ increased S by approximately 7% and 5%, respectively. The above cation effects reflected only alterations in the membrane fluidity and were not due to changes in probe–probe interactions. Ca²⁺ and La³⁺ at these concentrations decrease the activities of such plasma membrane enzymes as Na⁺, K⁺-ATPase and adenylyl cyclase, and it is suggested that the inhibition of these enzymes may be due in part to cation-mediated decreases in the lipid fluidity.     

Direct evidence for immiscible cholesterol domains in human ocular lens fiber cell plasma membranes.

The molecular structure of human ocular lens fiber cell plasma membranes was examined directly using small angle x-ray diffraction approaches. A distinct biochemical feature of these membranes is their high relative levels of free cholesterol; the mole ratio of cholesterol to phospholipid (C/P) measured in these membranes ranges from 1 to 4. The organization of cholesterol in this membrane system is not well understood, however. In this study, the structure of plasma membrane samples isolated from nuclear (3.3 C/P) and cortical (2.4 C/P) regions of human lenses was evaluated with x-ray diffraction approaches. Meridional diffraction patterns obtained from the oriented membrane samples demonstrated the presence of an immiscible cholesterol domain with a unit cell periodicity of 34.0 A, consistent with a cholesterol monohydrate bilayer. The dimensions of the sterol-rich domains remained constant over a broad range of temperatures (5-20 degrees C) and relative humidity levels (31-97%). In contrast, dimensions of the surrounding sterol-poor phase were significantly affected by experimental conditions. Similar structural features were observed in membranes reconstituted from fiber cell plasma membrane lipid extracts. The results of this study indicate that the lens fiber cell plasma membrane is a complex structure consisting of separate sterol-rich and -poor domains. Maintenance of these separate domains may be required for the normal function of lens fiber cell plasma membrane and may interfere with the cataractogenic aggregation of soluble lens proteins at the membrane surface.     

Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.

Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS.     

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.     

Cholesterol content drives distinct pharmacological behaviours of micro-opioid receptor in different microdomains of the CHO plasma membrane

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human micro-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.     

Cholesterol content drives distinct pharmacological behaviours of µ-opioid receptor in different microdomains of the CHO plasma membrane

Cholesterol in the plasma membrane of eukaryotic cells contributes to modulating the functions and signalling pathways of numerous transmembrane proteins, including G protein Coupled Receptors (GPCRs). We have previously shown that the function of the human µ-opioid receptor (hMOR) expressed in Saccharomyces cerevisiae is modulated by sterols including cholesterol. Here, we investigated the effects of cholesterol content on hMOR pharmacology and on hMOR partitioning in cholesterol-poor and -rich domains in eukaryotic mammalian cells (CHO). We show that cholesterol is required for the stabilization of a receptor conformation with high agonist affinity and for triggering G-protein activation after agonist binding to the receptor. Biochemical analysis of untreated and cholesterol-depleted membranes in cells expressing hMOR indicated that the receptor is only present in cholesterol poor domains, in the basal state. After agonist binding to untreated CHO membranes, two distinct populations of receptor were found in cholesterol-rich and -poor domains. Cholesterol depletion or treatment of CHO membranes with the G-protein-decoupling agent GppNHp prevented the redistribution, indicating that receptor activated states localized into cholesterol-rich domains. Pharmacological data and biochemical analysis indicate that distinct activated conformations of hMOR exist in CHO plasma membrane and correspond to microdomains differing by thickness and proportions of lipid components, including cholesterol.     

Effects of temperature and benzyl alcohol on the structure and adenylate cyclase activity of plasma membranes from bovine adrenal cortex

Adenylate cyclase activation by corticotropin (ACTH), fluoride and forskolin was studied as a function of membrane structure in plasma membranes from bovine adrenal cortex. The composition of these membranes was characterized by a very low cholesterol and sphingomyelin content and a high protein content. The fluorescent probes 1,6-diphenylhexa-1,3,5-triene (DPH) and a cationic analogue 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) were, respectively, used to probe the hydrophobic and polar head regions of the bilayer. When both probes were embedded either in the plasma membranes or in liposomes obtained from their lipid extracts, they exhibited lifetime heterogeneity, and in terms of the order parameter S, hindered motion. Under all the experimental conditions tested, S was higher for TMA-DPH than for DPH but both S values decreased linearly with temperature within the range of 10 to 40 degrees C, in the plasma membranes and the liposomes. This indicated the absence of lipid phase transition and phase separation. Addition to the membranes of up to 100 mM benzyl alcohol at 20 degrees C also resulted in a linear decrease in S values. Membrane perturbations by temperature changes or benzyl alcohol treatment made it possible to distinguish between the characteristics of adenylate cyclase activation with each of the three effectors used. Linear Arrhenius plots showed that when adenylate cyclase activity was stimulated by forskolin or NaF, the activation energy was similar (70 kJ.mol-1). Fluidification of the membrane with benzyl alcohol concentrations of up to 100 mM at 12 or 24 degrees C produced a linear decrease in the forskolin-stimulated activity, that led to its inhibition by 50%. By contrast, NaF stabilized adenylate cyclase activity against the perturbations induced by benzyl alcohol at both temperatures. In the presence of ACTH, biphasic Arrhenius plots were characterized by a well-defined break at 18 degrees C, which shifted at 12.5 degrees C in the presence of 40 mM benzyl alcohol. These plots suggested that ACTH-sensitive adenylate cyclase exists in two different states. This hypothesis was supported by the striking difference in the effects of benzyl alcohol perturbation when experiments were performed below and above the break temperature. The present results are consistent with the possibility that clusters of ACTH receptors form in the membrane as a function of temperature and/or lipid phase fluidity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cholesterol dynamics in membranes.

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.     

Effects of cholesterol on lipid organization in human erythrocyte membrane

The molar ratio of cholesterol to phospholipid (C/P) in human erythrocyte membrane is modified by incubating the cells with liposomes of various C/P ratios. The observed increase in cell surface area may be accounted for by the addition of cholesterol molecules. Fusion between liposomes and cells or attachment of liposomes to cells is not a significant factor in the alteration of C/P ratio. Onset temperatures for lipid phase separation in modified membranes are measured by electron diffraction. The onset temperature increases with decreasing C/P ration from 2 degrees C at C/P = 0.95 to 20 degrees C at C/P = 0.5. Redistribution of intramembrane particles is observed in membranes freeze-quenched from temperatures below the onset temperature. The heterogeneous distribution of intramembrane particles below the onset temperature suggests phase separation of lipid, with concomitant segregation of intramembrane protein into domains, even in the presence of an intact spectrin network.     

5'-Nucleotidase is activated upon cholesterol-depletion of liver plasma membranes

The cholesterol content of rat liver plasma membranes was manipulated using either cholesterol-free or cholesterol-enriched liposomes. Removal of cholesterol from the membranes led to a marked increase in 5'-nucleotidase activity. However, increase in cholesterol content failed to exert any significant effect on 5'-nucleotidase activity. Arrhenius plots of the activity of the native enzyme exhibited a break at around 28 degrees C with the activation energy of the reaction less above this temperature than below. In cholesterol-depleted membranes a single break at around 26 degrees C was observed with activation energies greater above this temperature than below it. In cholesterol-enriched membranes Arrhenius plots were linear over the range examined. It is suggested that the lipid environment of the external half of the bilayer only influences 5'-nucleotidase activity in these membranes and that cholesterol exerts controlling effects on both the activity and conformation of the enzyme in native membranes.     

Effects of calcium,lanthanum, and temperature on the fluidity of spin-labeled human platelets

Summary Previous platelet studies have shown that calcium plays important roles in stimulus-secretion coupling, aggregation, and other membrane-associated functions. In addition, lanthanum induces platelet aggregation and the platelet release reaction and also influences platelet responsiveness to various stimuli. The spin-label results presented here suggest that one mechanism through which calcium and lanthanum mediate their effects on platelet functions may be by decreasing the lipid fluidity of the surface membrane.The structure of platelet membrane lipids was examined with the spin-label method. Washed human platelets were labeled with the 5-, 12- and 16-nitroxide stearic acid spin probes. Order parameters which measure the fluidity of the lipid environment of the incorporated probe may be calculated from the electron spin resonance (ESR) spectra of 5-nitroxide stearate [I(12,3)]-labeled cells. Evidence is presented which indicates that these spectra principally reflect properties of the platelet surface membrane lipids. The membrane fluidity increased with temperature for the range 17 to 37 °C. Either calcium or lanthanum additions to intact cells increased the rigidity of the platelet membranes at 37 °C, although the La³⁺ effect was larger and occurred at lower concentrations than that of Ca²⁺. For example, addition of 1mm La³⁺ or 4mm Ca²⁺ increased the order parameter of I(12,3)-labeled platelets by 4.3±1.7% or 2.1±0.5%. Preliminary studies conducted on purified platelet plasma membranes labeled with I(12,3) indicated that 1mm LaCl₃ or 4mm CaCl₂ additions similarly decreased the lipid fluidity at 37 °C. The above cation-induced effects on the fluidity of whole platelets were reversed by the use of the divalent cation-chelating agent ethylene glycol-bis-(-aminoethyl ether)-N,N-tetra-acetic acid (EGTA). Lastly, lanthanum (0.2–1mm) caused rapid aggregation of platelets which were suspended in a 50-mm Tris buffer pH 7.4 that did not contain adenosine.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.