Interaction of the chemopreventive agent resveratrol and its metabolite,piceatannol, with model membranes

Resveratrol and piceatannol are plant-derived polyphenols possessing extremely wide range of biological activities such as cancer chemopreventive, cardio- and neuroprotective, antioxidant, anti-inflammatory, anticancer and lifespan extending properties. Despite great interest in these stilbenes, their interactions with lipid bilayers have not been extensively studied. In the present work, the interaction of both resveratrol and piceatannol with model membranes composed of phosphatidylcholine (DMPC and DPPC) was investigated by means of fluorescence spectroscopy, differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR). Generalized polarization of two fluorescent probes Laurdan and Prodan measured in pure lipid and lipid:stilbene mixtures revealed that resveratrol and piceatannol changed bilayer properties in both gel-like and liquid crystalline phase and interacted with lipid headgroup region of the membrane. These findings were corroborated by DSC experiments in which the stilbene-induced decrease of lipid melting temperature and transition cooperativity were recorded. Resveratrol and piceatannol restricted also the ESR-measured mobility of spin probes GluSIN18, 5DSA and 16DSA with nitroxide group localized at different depths. Since the most pronounced effect was exerted on the spin probe located near membrane surface, we concluded that also ESR results pointed to the preferential interaction of resveratrol and piceatannol with headgroup region of lipid bilayer.     

Use of fluorescence probes 1-aniline-8-naphthalene sulfonate and pyrene for studying the localisation of proteins in inner membranes from wheat etioplasts

The fluorescence probes 1-aniline-8-naphthalene sulfonate (ANS) and pyrene were applied for characterisation of the light-induced changes in etioplast inner membranes (EPIMs) from 7 d-old dark-grown wheat seedlings (Triticum aestivum L. cv. Pobeda). The major aim was to obtain information about the localisation of membrane proteins in the EPIMs, using probes situated in different regions of the membranes. The quenching of tryptophan fluorescence showed tha the main parts of proteins were accessible to the pyrene buried in the lipid bilayer which suggests that most of the proteins also enter the lipid bilayer. The substantial quenching of the tryptophan fluorescence by the surface-situated ANS demonstrated that a part of the tryptophan residues was probably localised close to the membrane surface. The registered changes after irradiation could be explained by the presence of large aggregates of NADPH-protochlorophyllide oxidoreductase (POR), protochlorophyllide (PChlide) and NADPH in membranes that start to disconnect and redistribute along the prothylakoids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

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.     

Fluorescence studies of the blood platelet membranes associated with fibrinogen

To follow microviscosity changes in membranes associated with fibrinogen binding to human platelets, specific fluorescent probes were used and their fluorescence anisotropy was analysed. The degree of fluorescence anisotropy of diphenylhexatriene, anilinonaphthalene sulfonate (ANS) and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Fluorescence polarization analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase in the membrane lipid rigidity. On the other hand, changes in the fluorescence anisotropy of membrane tryptophans and N-(3-pyrene)maleimide suggest augmented mobility of the membrane proteins. The binding of fibrinogen to the membrane receptors is not accompanied by any change in the fluorescence intensity of ANS attached to the membranes. This may suggest that covering of platelets with fibrinogen molecules does not influence the surface membrane charge.     

ESR studies of the erythrocyte membrane skeletal protein network: influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

Microenvironment changes of human blood platelet membranes associated with fibrinogen binding

Alterations in the membrane organization caused by fibrinogen binding to human blood platelets and their isolated membranes were analyzed by fluorescence and electron spin resonance measurements. The degree of fluorescent anisotropy of DPH, ANS and fluorescamine increased significantly when fibrinogen reacted with its membrane receptors. Both fluorescence and ESR analyses showed that fibrinogen binding to platelet membranes is accompanied by an increase of the membrane lipid rigidity. This effect seems to be indirect in nature and is mediated by altered membrane protein interactions. As it has been shown that an increased membrane lipid rigidity leads to a greater exposure of membrane proteins, including fibrinogen receptors, this might facilitate a formation of molecular linkages between neighboring platelets. On the other hand, changes of fluorescence anisotropy of membrane tryptophans and N-(3-pyrene) maleimide suggest the augmented mobility of the membrane proteins. Evidence is presented which indicated that the binding of fibrinogen to the membrane receptors is not accompanied by any changes in the fluorescence intensity of ANS attached to the membranes. It may suggest that the covering of platelets with fibrinogen does not influence the surface membrane charge. In contrast to fibrinogen, calcium ions caused an increase of the fluorescence intensity resulting from the more efficient binding of ANS to the platelet membranes.     

ESR studies of the erythrocyte membrane skeletal protein network: Influence of the state of aggregation of spectrin on the physical state of membrane proteins, bilayer lipids, and cell surface carbohydrates

The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bis-phosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P < 0.002 and P < 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding sites is observed (P < 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P < 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network.     

Reliability of nitroxide spin probes in reporting membrane properties: a comparison of nitroxide- and deuterium-labeled steroids

The reliability for the study of membrane properties of the steroid nitroxide spin probe, 3-doxylcholestane, was tested by comparison of analogous data for the deuterated steroid, cholesterol-3 alpha-d. Good agreement between the two probes was found for the dependence of their order parameters on variation of temperature or cholesterol concentration in egg phosphatidylcholine bilayers. This finding is contrasted with the results of a previous study of fatty acid probes where poor agreement was found for the spectral responses of nitroxide- and deuterium-labeled species. The angular dependence of the ESR spectra of nitroxide-labeled probes in oriented multibilayer films was examined to determine if the probes were oriented in a tilted fashion in the bilayer. The 3-doxylcholestane probe and a doxylstearic acid labeled at position 14 orient with their long molecular axes perpendicular to the bilayer plane. In contrast, the stearic acid probe nitroxide labeled at position 5 does not appear to orient in such a fashion. We suggest that the behavior of the latter probe reflects the difficulty of inserting a bulky nitroxide group into a highly ordered region of the bilayer rather than an inherent tilting of the phospholipid acyl chains. On the basis of the comparisons between various types of probes, some suggestions are made concerning the choice of ESR spin probe to obtain reliable information in membrane studies.     

Changes in the lipid physical state in human erythrocyte membranes subjected lead exposure in vitro

The effect of lead acetate on the physical state of membrane lipids in human erythrocytes in vitro was studied using the lipophilic fluorescence probe 1,6-diphenyl-1,3,5-hexatriene and spin probes 16-doxyl-stearate and iminoxyl palmitic acid. It was shown that 2-10 microM lead acetate causes an increase in both intensity and polarization of fluorescence of 1,6-diphenyl-1,3,5-hexatriene, indicating changes in the microviscosity of the lipid bilayer of erythrocyte membranes. Judging from the parameters of EPR spectra of 16-doxyl stearate and iminoxyl palmitic acid incorporated into erythrocyte membranes, 2-10 microM lead acetate increases the heterogeneity of the lipid bilayer in surface and deep hydrophobic layers of the erythrocyte membrane.     

Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

We derive equations that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of electron spin resonance (ESR) nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. We make use of an assumption used by us in an earlier paper. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. We consider cases where the bilayer is either in a single homogeneous phase or in a two-phase region. We apply our equations to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase, both for T greater than Tc. The following conclusions were derived: (i) Ca2+-ATPase oligomers are "randomly" distributed, for the concentrations studied, in the fluid phase. (ii) There is no fixed stoichiometric ratio of "boundary" lipids and oligomers. (iii) Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. Finally, we apply our equations to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T less than Tc. We find that the results reported are in accord with the predictions of the model. In the Appendix, we show that an analytical expression for probabilities used by us is in very good agreement with the results of computer simulation.     

Probing Orientational Behavior of MHC Class I Protein and Lipid Probes in Cell Membranes by Fluorescence Polarization-Resolved Imaging

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.     

没食子酸丙酯和没食子酸异丁酯对人红细胞膜结构与功能的影响

本文应用荧光探剂ANS(1—苯胺—8萘磺酸)、NPN(N—苯基—1—萘胺)和DPH(1.6—二苯基—1.3.5—已三烯)观察没食子酸丙醋和没食子酸异丁酯对人红细胞膜流动性和相变温度以及Na~ -K~ ATP酶活性的影响.实验结果指出该两种化合物均能:(1)降低与膜结合的荧光探剂强度但不改变探剂在水相与膜相的分配比例:(2)降低膜脂的相变温度,增加膜的流动性;(3)抑制红细胞膜Na~ -K~ ATP酶活性;(4)标记红细胞膜的DPH偏振度随化合物浓度的增加而降低,膜的流动性增加.在给定的浓度范围内,两种化合物的效应表现为明显的量效关系与构效关系.从上述结果推测该两种化合物可能是通过改变膜脂结构、膜蛋白的脂类环境而调节膜的功能,成为其治疗疾病的机理之一.     

Melittin Stimulates Incorporation and Degradation of Sphingomyelin in Synaptosomal Plasma Membranes

Abstract: Melittin enhanced sphingomyelin (SPM) degradation by the neutral membrane-bound sphingomyelinase from calf brain synaptosomal plasma membranes (SYM) up to 20-fold. Melittin in concentrations as high as 100 μM did not significantly alter membrane fluidity of SYM as measured by fluorescence depolarization and electron spin resonance (ESR) using diphenylhexatriene and a doxy1 derivative of SPM, respectively. In the concentration range 100-1000 μM. melittin was observed to rigidify SYM. The incorporation of SPM.erivatives into the lipid bilayer of SYM.as demonstrated by ESR measurements. Melittin enhanced the uptake of SPM-derivatives into SYM.     

膜融合剂对脂质体及血影膜膜流动性的影响

本文用荧光探针ANS,DPH与A研究了几种膜融合剂对脂质体与血影膜流动性的影响.蔗糖使PS脂质体的脂双层流动性降低,探针越是在极性区流动性越小,说明蔗糖主要作用于脂双层的极性区;蔗糖也使血影膜流动性降低,此作用是可逆的.油酸甘油脂(GMO)使PS脂质体的流动性增加,且越是在疏水区内部,流动性增加得越大,说明GMO主要是作用于脂双层的非极性区:GMO也使血影膜流动性增加,此作用是不可逆的.二甲亚砜(DMSO)对血影膜的作用,两种不同荧光探针不一样,对DPH的作用出现双相让,低浓度与高浓度的作用结果分别与蔗糖和GMO的作用一致.     

Calcium and chlorpromazine interactions in rat synaptic plasma membranes. A spin-label and fluorescence probe study.

The effects of calcium and of the psychoactive drug chlorpromazine (CPZ) on the rat synaptic plasma membrane have been studied using two stearic nitroxide spin labels having their doxyl groups in positions 5 and 16 and the fluorescent probe 1-anilinonaphtalene-8-sulfonate (ANS). The mobility of the 5-doxyl stearic spin label which probes the membrane phospholipids in the vicinity of their polar heads is decreased in the presence of both compounds. Calcium is more efficient in this respect than CPZ. In spite of this qualitative similarity of action, CPZ inhibits the effect of calcium and vice versa. No modification of the 16-doxyl stearic spectrum has been observed even at high calcium or CPZ concentrations. An increase in fluorescence intensity and a blue shift in the emission wavelength of ANS-probed membranes are observed with very low CPZ concentrations (10^?7 to 10^?5m). With higher concentrations, a further intensity increase and a further blue shift are due to direct interaction between ANS and CPZ. Calcium also increases the fluorescence intensity of ANS-labeled membranes in the concentration range 10^?5–10^?2m. As for the spin-label data, the effects of both compounds are mutually competitive. It is concluded that calcium interacts principally with the phospholipid polar heads of this type of membrane. However, the competition with CPZ suggests indirectly that this ion is also bound to membrane proteins. CPZ has an affinity for membrane lipids only at high concentrations. In its pharmacologically active concentration range, it is located preferentially on the membrane proteins.     

Fluorescence Studies of Red Blood Cell Membranes from Individuals with Huntington''s Disease

Fluorescence spectroscopic methods were used to investigate and compare the properties of erythrocyte membranes from individuals with Huntington's Disease (HD) and from normal individuals. Erythrocyte ghosts were labeled with four different fluorescent probes: 1,6-diphenylhexatriene (DPH); 6-lauroyl-2-(dimethylamino)-naphthalene (Laurdan); 2-(4-maleimide anilino)-naphthalene-6-sulfonic acid (MIANS) and 5-(iodoacetamidoethyl)aminoaphthalene-1-sulfonic acid (IAEDANS). DPH is sensitive to the microviscosity of the membranes. Laurdan is highly sensitive to the polarity and relaxation time of its environment. MIANS and IAEDANS both react covalently with sulfhydryl groups in membrane proteins. Erythrocyte membranes were labeled separately with each of these four probes, and we measured the centers of gravity of the fluorescence emission, the fluorescence anisotropies, and the fluorescence lifetimes. In 10 separate experiments, including a total of 24 patients and 14 control individuals, we found no significant differences between the two groups for any of the probes or spectral parameters. These results do not support the existence of a "generalized membrane defect" in individuals with HD.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

Electron spin resonance and biochemical studies of the interaction of the polyamine, spermine, with the skeletal network of proteins in human erythrocyte membranes

Spermine (N, N'-bis(aminopropyl)-1,4-butanediamine) is a polyamine thought to be important in several cell regulatory processes. Previous studies had shown that spermine prevented the lateral diffusion of transmembrane proteins in human erythrocyte ghosts (Schindler et al. (1980) Proc. Natl. Acad. Sci. USA 77, 1457-1461). In this paper, we present results of studies on the effect of spermine on erythrocyte membranes by employing electron spin resonance spin-labeling techniques in conjunction with spin labels specific for skeletal proteins, bilayer lipids or cell-surface sialic acid of the membrane and by employing SDS-polyacrylamide gel electrophoresis analysis of extracted spectrin and Triton shells. The major findings are: (1) spermine significantly decreases the segmental motion of protein spin-label binding sites (P less than 0.0001), which are predominantly on cytoskeletal proteins; (2) addition of spermine leads to a significant increase in the rotational motion of spin-labeled terminal sialic acid residues (P less than 0.001), most of which are located on glycophorin A, a result which may be secondarily caused by spermine-induced aggregation of cytoskeletal proteins and the cytoplasmic pole of this transmembrane sialoglycoprotein; (3) spermine completely inhibits the low-ionic strength extraction of spectrin, the major protein of the skeletal network which is attached to the bilayer proteins by two or more connecting proteins; (4) pretreatment of ghosts with spermine followed by Triton extraction resulted in the retention of significantly increased amounts of Band 3 and other skeletal and bilayer proteins including Bands 4.2, 6 and 7 in Triton X-100 shells relative to that of control-treated ghosts. These results suggest that spermine acts both to increase protein-protein interactions in the cytoskeletal protein network and to bridge skeletal and bilayer proteins and are discussed with reference to possible molecular mechanisms by which spermine may influence cell functions.     

Effects of K+-depolarization on the physical state of membrane lipids in brain synaptosomes of the rat

The microstructure of lipid bilayer in synaptosomes from rat brain upon K+-depolarization (30 mM) was studied using the inductive resonance energy transfer (IRET) from proteins to the fluorescent probes, pyrene and DMC (4-dimethylaminochalcone). The effectiveness of IRET was not changed by the K+-depolarization. The monomer-to-eximer ration (Fm285/Fe285) of pyrene fluorescence intensities in IRET was 1.5 times lower upon depolarization than in controls. This suggested a decreased microviscosity of the lipid bilayer in immediate environment to proteins of the synaptosomal membrane. The Fm338/Fee338 ratio as well as polarization of DMC fluorescence indicative of the bulk lipid phase were not altered under these conditions. Neither cytochalasin B not colchicine had any effect on fluorescence polarization of DMC both in control and depolarized synaptosomes. It is suggested that the increased lateral mobility of protein-associated lipid molecules found in depolarized synaptosomes may be caused by alterations in the activity of ion channels and ion pumps or by restructuring of the cytoskeletal network.