Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.     

Organization of lipids in sarcoplasmic reticulum membrane and Ca2+-dependent ATPase activity.

The organization of lipids in sarcoplasmic reticulum membrane was studied with a variety of stearic spin labels and a phosphatidylcholine spin label. The ESR spectra of the spin-labeled membranes consisted of two components, one due to labels in lipid bilayer structure and the other due to more immobilized labels. The relative intensity of the immobilized component increased when the lipid content of the membrane was decreased by treatment with phospholipase A [EC 3.1.1.4] and subsequent washing with bovine serum albumin. Membrane containing 30% of the intact phospholipid, i.e.0.15 mg of phospholipid per mg of protein, showed a spectrum consisting only of the immobilized component (the overall splitting ranged from 58.5 G to 60.5 G). The immobilized component was ascribed to lipids complexed with protein. The fraction of lipids in the two different organizations was determined from the ESR spectrum. The activity of the Ca2+-Mg2+ dependent ATPase [ATP phosphohydrolase, EC 3.6.1.3] was found to increase almost linearly with the lipid bilayer content in the membrane, whereas phosphoenzyme formation was almost independent of the bilayer content. This indicated that the bilayer structure is necessary for the ATPase to attain its full transport activity.     

Bilayer structure in phospholipid-cytochrome c model membranes.

Lipid protein interactions in biological membranes differ markedly depending on whether the protein is intrinsic or extrinsic. These interactions are studied using lipid spin labels diffused into model systems consisting of phospholipid bilayers and a specific protein. Recently, an intrinsic protein complex, cytochrome oxidase, was examined and the data suggest there is a boundary layer of immobilized lipid between the hydrophobic protein surfaces and adjacent fluid bilayer regions. In the present study, a typical extrinsic protein, cytochrome c, was complexed with a cardiolipin/lecithin (1:4 by weight) mixture. The phospholipids in the presence and absence of cytochrome c exhibit typical bilayer behavior as jedged by four spin-labeling criteria: fluidity gradient, spectral anisotropy of oriented bilayers, response to hydration and the polarity profile. Any effects of cytochrome c on the ESR spectra of lipid spin labels are small, in contrast to the effects of intrinsic proteins. These data are consistent with electrostatic binding of cytochrome c to the charged groups of the phospholipids, and indicate that the presence of extrinsic proteins will not interfere with measurements of boundary lipid in intact biological membranes.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Phosphatidylcholine exchange between the boundary lipid and bilayer domains in cytochrome oxidase containing membranes.

A phospholipid spin label, 16-doxylphosphatidylcholine, is employed in a study of lipid--protein interactions in cytochrome oxidase containing membranes. Two methods are used to label the membranous cytochrome oxidase: dispersion in cholate with subsequent detergent removal, and fusion with vesicles of the pure phospholipid label in the absence of detergent. A fraction of the label is immobilized, which is calculated to fall in the range of 0.17--0.21 mg of phospholipid/mg of protein (0.15--0.19 after correction for lipids not extracted by chloroform--methanol). This narrow range of values is independent of methods of labeling, protein isolation, and lipid depletion within experimental error. When labeling by fusion is utilized, the patches of pure phosphatidylcholine spin label diffuse in the plane of the bilayer, become diluted, and demonstrate exchange with bound phospholipid. These observations are evidence that boundary lipid, as reflected by the partitioning of the phosphatidylcholine label, is in equilibrium with adjacent bilayer regions and that it consists of a relatively constant amount of phospholipid associated with the hydrophobic portion of the protein.     

Modulation of the catalytic properties of alpha-chymotrypsin by chemical modification at Tyr 146

XC Sarcoma, Vero and Aedes aegypti plasma membranes have been studied in viable cells and in purified membrane of XC Sarcoma cells by the spin label method. The temperature dependence of the order parameter of fatty acid spin labels is found to be linear in all three cells and membrane and shows no evidence of a lipid phase transition. The order parameter of the fatty acid labels substituted at the 5-position is shown to increase as a function of the cholesterol: phospholipid molar ratio in cells that have been studied to date. Cells attached to their growing surface are studied for the first time by electron paramagnetic resonance spectroscopy (EPR). The resulting spectra are anisotropic due to the non-spherical shape of the cells and show that these labels orient preferentially perpendicular to the cell surface. The viscosity of the extracted XC cell membrane is estimated to be 2.5 P from rotational correlation time measurements of the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO).     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Spin labelling study of interfacial properties of egg-phosphatidylcholine liposomes as a function of cholesterol concentrations

In order to gain insight into interfacial properties of liposomes composed of egg-phosphatidylcholine (egg-PC) and dihexadecyl-phosphate (DHP) as a function of 0, 8, 15, 29, 38, 45 mol% of cholesterol, dynamic properties of two long-chain spin labels: TEMPO-stearate (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl)-octa-decanoate) and TEMPO-stearamide (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl)-octa-decanamide) were studied by CW-ESR spectroscopy. These spin labels reflect motional properties in the region of phospholipid head-groups.Two different environments of TEMPO-stearate were determined at 29, 38 and 45 mol% of cholesterol. In the newly formed domain above 29 mol%, N-O moiety of the spin label was surrounded by larger amount of bound water and experienced slower motion than in the cholesterol poor domain. The fraction of the second more hydrophilic environment of the spin label increased with cholesterol concentration. TEMPO-stearamide, a hydrogen-bond donor, reported more polar environment and slower motion than TEMPO-stearate even in the absence of cholesterol. Only one spin label environment was determined for all cholesterol concentrations. Slowing down of the TEMPO-stearamide motion was obtained even at 8 mol% of cholesterol.     

Magnetic resonance studies of eukaryotic cells. III. Spin labeled fatty acids in the plasma membrane

XC Sarcoma, Vero and Aedes aegypti plasma membranes have been studied in viable cells and in purified membrane of XC Sarcoma cells by the spin label method. The temperature dependence of the order parameter of fatty acid spin labels is found to be linear in all three cells and membrane and shows no evidence of a lipid phase transition. The order parameter of the fatty acid labels substituted at the 5-position is shown to increase as a function of the cholesterol: phospholipid molar ratio in cells that have been studied to date. Cells attached to their growing surface are studied for the first time by electron paramagnetic resonance spectroscopy (EPR). The resulting spectra are anisotropic due to the non-spherical shape of the cells and show that these labels orient preferentially perpendicular to the cell surface. The viscosity of the extracted XC cell membrane is estimated to be 2.5 P from rotational correlation time measurements of the spin label 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO).     

The immiscible cholesterol bilayer domain exists as an integral part of phospholipid bilayer membranes

Electron paramagnetic resonance (EPR) spin-labeling methods were used to study the organization of cholesterol and phospholipids in membranes formed from Chol/POPS (cholesterol/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine) mixtures, with mixing ratios from 0 to 3. It was confirmed using the discrimination by oxygen transport and polar relaxation agent accessibility methods that the immiscible cholesterol bilayer domain (CBD) was present in all of the suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST) in the POPS membrane. The behavior of phospholipid molecules was monitored with phospholipid analogue spin labels (n-PCs), and the behavior of cholesterol was monitored with the cholesterol analogue spin labels CSL and ASL. Results indicated that phospholipid and cholesterol mixtures can form a membrane suspension up to a mixing ratio of ~2. Additionally, EPR spectra for n-PC, ASL, and CSL indicated that both phospholipids and cholesterol exist in these suspensions in the lipid-bilayer-like structures. EPR spectral characteristics of n-PCs (spin labels located in the phospholipid cholesterol bilayer, outside the CBD) change with increase in the cholesterol content up to and beyond the CST. These results present strong evidence that the CBD forms an integral part of the phospholipid bilayer when formed from a Chol/POPS mixture up to a mixing ratio of ~2. Interestingly, CSL in cholesterol alone (without phospholipids) when suspended in buffer does not detect formation of bilayer-like structures. A broad, single-line EPR signal is given, similar to that obtained for the dry film of cholesterol before addition of the buffer. This broad, single-line signal is also observed in suspensions formed for Chol/POPS mixtures (as a background signal) when the Chol/POPS ratio is much greater than 3. It is suggested that the EPR spin-labeling approach can discriminate and characterize the fraction of cholesterol that forms the CBD within the phospholipid bilayer.     

Spin-label studies on phosphatidylcholine-cholesterol membranes: effects of alkyl chain length and unsaturation in the fluid phase

Dynamic properties of phosphatidylcholine-cholesterol membranes in the fluid phase and water accessibility to the membranes have been studied as a function of phospholipid alkyl chain length, saturation, mole fraction of cholesterol, and temperature by using spin and fluorescence labelling methods. The results are the following: (1) The effect of cholesterol on motional freedom of 5-doxyl stearic acid spin label (5-SASL) and 16-doxyl stearic acid spin label (16-SASL) in saturated phosphatidylcholine membrane is significantly larger than the effects of alkyl chain length and introduction of unsaturation in the alkyl chain. (2) Variation of alkyl chain length of saturated phospholipids does not alter the effects of cholesterol except in the case of dilauroylphosphatidylcholine, which possesses the shortest alkyl chains (12 carbons) used in this work. (3) Unsaturation of the alkyl chains greatly reduces the ordering effect of cholesterol at C-5 and C-16 positions although unsaturation alone gives only minor fluidizing effects. (4) Introduction of 30 mol% cholesterol to dimyristoylphosphatidylcholine membranes decreases the lateral diffusion constants of lipids by a factor of four, while it causes only a slight decrease of lateral diffusion in dioleoylphosphatidylcholine membranes. (5) If compared at the same temperature, 5-SASL mobilities plotted as a function of mole fraction of cholesterol in the fluid phases of dimyristoylphosphatidylcholine-, dipalmitoylphosphatidylcholine- and distearoylphosphatidylcholine-cholesterol membranes are similar in wide ranges of temperature (45-82 degrees C) and cholesterol mole fraction (0-50%). (6) In isothermal experiments with saturated phosphatidylcholine membranes, 5-SASL is maximally immobilized at the phase boundary between Regions I and III reported by other workers (Recktenwald, D.J. and McConnell, H.M. (1981) Biochemistry 20, 4505-4510) and becomes more mobile away from the boundary in Regions I and III. (7) 5-SASL in unsaturated phosphatidylcholine membranes showed a gradual monotonic immobilization with increase of cholesterol mole fraction without showing any maximum in the range of cholesterol fractions studied. (8) By rigorously determining rigid-limit magnetic parameters of cholestane spin labels in membranes from Q-band second-derivative ESR spectra to monitor the dielectric environment around the nitroxide radical, it is concluded that cholesterol incorporation increases water accessibility in the hydrophilic loci of the membrane. In contrast, 12-(9-anthroyloxy)stearic acid fluorescence showed that water accessibility is decreased in the hydrophobic loci of the membrane.     

A spin-label study of protein-lipid interaction in sarcoplasmic reticulum of rabbit skeletal muscle

Whether or not the thermotropic change at about 18 degrees C in the physical state of Ca2+-ATPase protein molecules of sarcoplasmic reticulum membranes could be transmitted to lipids through protein-lipid interactions was investigated using a spin-label technique. Fatty acid spin labels were used to probe the bulk membrane lipids while long-chain spin labels attached at one end to the Ca2+-ATPase molecules through a covalent bond were used to monitor the boundary lipids. The results on the temperature-dependence of alkyl-chain flexibility of lipid molecules indicate that the change in the state of the protein molecules is accompanied by one of the boundary lipids, but not of the bulk lipids.     

Lipid protein interactions in mitochondria. Spin and fluorescence probe studies on the effect of n-alkanols on phospholipid vesicles and mitochondrial membranes.

The effect of n-butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n-alcohols is related to their hydrophobicity.Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data.As for the spin label data, the effect of the series of aliphatic n-alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.     

Lipid protein interactions in mitochondria. VIII. Effect of general anesthetics on the mobility of spin labels in lipid vesticles and mitochondrial membranes

We have studied the effect of general anesthetics on the mobility of two stearic acid spin labels (5-doxyl stearic acid and 16-doxyl stearic acid) in bovine heart mitochondria and in phospholipid vesicles made from either mitochondrial lipids or commercial soybean phospholipids. The general anesthetics used include nonpolar compounds (alcohols, halothane, pentrane, diethyl ether, chloroform) and the amphipathic compound, ketamine. All anesthetics tested increase the mobility of the spin labels in phospholipid vesicles to a limited extent up to a concentration where the ESR spectra become those of free spin labels. On the other hand, anesthetics have a pronounced effect on mitochondrial membranes at concentrations as low as those known to produce general anesthesia; the effect is lower near the bilayer surface (5-doxyl stearic acid) and very strong in the bilayer core (16-doxyl stearic acid). The effects of anesthetics are mimicked by the detergent, Triton X-100. We suggest that the discrepancy between the action of anesthetics in mobilizing the spin labels in lipid vesicles and in membranes results from labilization of lipid protein interactions.     

Liver ischemia increases the molecular order of microsomal membranes by increasing the cholesterol-to-phospholipid ratio

An accelerated degradation of phospholipid is the likely basis of irreversible cell injury in ischemia, and the membranes of the endoplasmic reticulum of the liver are a convenient system with which to study the effect of such a disturbance on the structure and function of cellular membranes. In the present report, electron spin resonance spectroscopy has been used to evaluate changes in the molecular ordering of microsomal membrane phospholipids in the attempt to relate the loss of lipid to alterations in membrane structure. The order parameter, S, was calculated from spectra reflecting the anisotropic motion of 12-doxyl stearic acid incorporated into normal and 3-h ischemic microsomal membranes. Over the temperature range 4-40 degrees C, the molecular order (S) of ischemic membranes was increased by 8-10%. This increase was reproduced in the ordering of the phospholipids in liposomes prepared from total lipid extracts of the same membranes. In contrast, after removal of the neutral lipids, liposomes prepared from phospholipids of ischemic and control membranes had the same molecular order. There were no differences in the phospholipid species of control and ischemic membranes or in the fatty acid composition of the phospholipids. In the neutral lipid fraction of ischemic membranes, however, triglycerides and cholesterol were increased compared to control preparations. There were no free fatty acids. The total cholesterol content of the liver was unchanged after 3 h of ischemia. The cholesterol-to-phospholipid ratio of ischemic membranes, however, was increased by 22% from 0.258 to 0.315 as a consequence of the loss of phospholipid. Addition of cholesterol to the control total lipid extracts to give a cholesterol-to-phospholipid ratio the same as in ischemic membranes resulted in liposomes with order parameters similar to those of liposomes prepared from ischemic total lipids. It is concluded that the degradation of the phospholipids of the microsomal membrane results in a relative increase in the cholesterol-to-phospholipid ratio. This is accompanied, in turn, by an increased molecular order of the residual membrane phospholipids.     

Lipid phase of transverse tubule membranes from skeletal muscle. An electron paramagnetic resonance study.

The lipid phase of transverse tubule membrane was probed with a variety of fatty acid spin labels. The motion of the probe increased as the distance between the spin label and polar head group increased, in agreement with results reported in other membranes. The value of the order parameter at 37 degrees C for a fatty acid spin label containing the label attached to its fifth carbon atom was closer to values reported for bacterial membranes than to the lower values reported for other mammalian membranes. Order parameters for spin labels containing the label nearer to the center of the bilayer were closer to the values reported in other mammalian membranes than to values reported for bacterial membranes. These results indicate that the lipid segments in the vicinity of the polar head group, and less so those near the center of the bilayer, are motionally more restricted in transverse tubules than in other mammalian membranes. In particular, the lipid phase of the transverse tubule membrane is less fluid than that of the sarcoplasmic reticulum membrane. A possible role of the high cholesterol content of transverse tubules in generating the lower fluidity of its lipid phase is discussed.     

Detergent resistant domains in erythrocyte membranes survive after cell cholesterol depletion: an EPR spin label study

We use electron paramagnetic resonance (EPR) with liposoluble spin labels in order to study the lipid structures obtained after Triton X-100 extraction of erythrocyte membranes. The apparent order profile in these detergent resistant membranes (DRM) is very similar to that of the parent membrane, although with higher absolute values, consistent with a liquid-ordered state. DRM could also be obtained from erythrocytes previously depleted in a 40% of their membrane cholesterol, in apparent opposition to the phenomenon of raft disruption reported by other authors. However, the protein profile of these samples showed important differences with that of DRM from untreated cells. The analysis of our results suggests that the effect of Triton X-100 on cholesterol depleted erythrocytes is limited to the solubilization of raft proteins, without disrupting the lipid matrix of DRM.     

Lipid rafts are enriched in arachidonic acid and plasmenylethanolamine and their composition is independent of caveolin-1 expression: a quantitative electrospray ionization/mass spectrometric analysis

Lipid rafts are specialized cholesterol-enriched membrane domains that participate in cellular signaling processes. Caveolae are related domains that become invaginated due to the presence of the structural protein, caveolin-1. In this paper, we use electrospray ionization mass spectrometry (ESI/MS) to quantitatively compare the phospholipids present in plasma membranes and nondetergent lipid rafts from caveolin-1-expressing and nonexpressing cells. Lipid rafts are enriched in cholesterol and sphingomyelin as compared to the plasma membrane fraction. Expression of caveolin-1 increases the amount of cholesterol recovered in the lipid raft fraction but does not affect the relative proportions of the various phospholipid classes. Surprisingly, ESI/MS demonstrated that lipid rafts are enriched in plasmenylethanolamines, particularly those containing arachidonic acid. While the total content of anionic phospholipids was similar in plasma membranes and nondetergent lipid rafts, the latter were highly enriched in phosphatidylserine but relatively depleted in phosphatidylinositol. Detergent-resistant membranes made from the same cells showed a higher cholesterol content than nondetergent lipid rafts but were depleted in anionic phospholipids. In addition, these detergent-resistant membranes were not enriched in arachidonic acid-containing ethanolamine plasmalogens. These data provide insight into the structure of lipid rafts and identify potential new roles for these domains in signal transduction.     

A comparative study of the composition of the microsomal membranes of the liver, brain and skeletal muscles in vertebrates]

Phospholipid and cholesterol amounts, intrinsic protein/lipid ratios in liver, brain and skeletal muscle microsomal membranes of 14 species of vertebrate animals have been studied. No significant differences between phospholipid amounts in tissues as well as vertebrate classes have been discovered. The highest cholesterol amount has been found in brain microsomes, the smallest one in sarcoplasmic reticulum membranes. In reptile brain and muscle microsomes a higher amount of cholesterol compared to that in species of other vertebrate classes has been found. In brain membranes intrinsic protein and lipid amounts are approximately equal, while in liver and muscle microsomes a protein component predominates. Phospholipid/protein ratio is larger in brain membranes than in liver and muscle ones. Cholesterol/protein ratio reaches the highest values in microsomal membranes of reptile tissues. Brain membranes of vertebrate animals are characterized by a greater stability of protein-lipid composition than liver and muscle ones.     

Plasma membrane lipid order and composition during adipocyte differentiation of 3T3F442A cells. Studies in intact cells with 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene

The plasma membrane lipid order of 3T3F442A cells was examined during the course of adipocyte differentiation by measuring the fluorescence polarization of 1-[4-(trimethylamino)phenyl]-6-phenylhexatriene. This cationic fluorophore labels the plasma membrane but does not rapidly redistribute to intracellular organellar membranes and can, therefore, be used to specifically probe the plasma membrane of intact cells. Studies with whole cells demonstrated that the plasma membrane of 3T3F442A cells becomes less ordered during the course of adipocyte conversion and that this alteration begins relatively early during the differentiation process. In addition, the lipid order of plasma membranes isolated from adipocyte-stage cells was found to be lower than the lipid order of the early, fibroblast-stage cells. Analysis of membrane lipid composition suggests that the molecular bases for the decrease in adipocyte plasma membrane lipid order are a large increase in the level of monounsaturated phospholipid acyl chains and a decrease in the molar ratio of cholesterol to phospholipid. The alteration in plasma membrane lipid composition may be specifically required for integral membrane protein function, since the differentiation-dependent fatty acid desaturase activity is known to be maintained even in the absence of triacylglycerol accumulation.