Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol.

d-Alpha-tocopherol (2R,4'R,8'R-Alpha-tocopherol) and d-alpha-tocotrienol are two vitamin E constituents having the same aromatic chromanol "head" but differing in their hydrocarbon "tail": tocopherol with a saturated and toctrienol with an unsaturated isoprenoid chain. d-Alpha-tocopherol has the highest vitamin E activity, while d-alpha-tocotrienol manifests only about 30% of this activity. Since vitamin E is considered to be physiologically the most important lipid-soluble chain-breaking antioxidant of membranes, we studied alpha-tocotrienol as compared to alpha-tocopherol under conditions which are important for their antioxidant function. d-Alpha-tocotrienol possesses 40-60 times higher antioxidant activity against (Fe2+ + ascorbate)- and (Fe2+ + NADPH)-induced lipid peroxidation in rat liver microsomal membranes and 6.5 times better protection of cytochrome P-450 against oxidative damage than d-alpha-tocopherol. To clarify the mechanisms responsible for the much higher antioxidant potency of d-alpha-tocotrienol compared to d-alpha-tocopherol, ESR studies were performed of recycling efficiency of the chromanols from their chromanoxyl radicals. 1H-NMR measurements of lipid molecular mobility in liposomes containing chromanols, and fluorescence measurements which reveal the uniformity of distribution (clusterizations) of chromanols in the lipid bilayer. From the results, we concluded that this higher antioxidant potency of d-alpha-tocotrienol is due to the combined effects of three properties exhibited by d-alpha-tocotrienol as compared to d-alpha-tocopherol: (i) its higher recycling efficiency from chromanoxyl radicals, (ii) its more uniform distribution in membrane bilayer, and (iii) its stronger disordering of membrane lipids which makes interaction of chromanols with lipid radicals more efficient. The data presented show that there is a considerable discrepancy between the relative in vitro antioxidant activity of d-alpha-tocopherol and d-alpha-tocotrienol with the conventional bioassays of their vitamin activity.     

The role of isoprenoid chain of alpha-tocopherol in the protection of synaptosomes against lipid peroxidation and phospholipase A2-induced damage

The role of the alpha-tocopherol molecule isoprenoid chain in synaptosomal membrane protection from lipid peroxidation activation and phospholipase A2 damage was investigated. A comparative study of alpha-tocopherol analogs differing in the length of the isoprenoid chain revealed that the increase in the chain length results in a decrease of the efficiency of inhibition in the course of synaptosomal lipid peroxidation activation. This effect is due to the diminution of mobility of chromanols in the lipid bilayer which is associated with an increase in the length of the isoprenoid fragment. The decreased efficiency of lipid peroxidation inhibition resulting from the lengthening of the chromanol nucleus phytol chain is concomitant with the appearance of new stabilizing properties, e. g., the ability to protect synaptosomal membranes from the damaging action of phospholipase A2. This effect is lost with a decrease in the length of the chromanol isoprenoid chain.     

Differential regulation of the lateral mobility of plasma membrane phospholipids by the extracellular matrix and cholesterol

In this study, we compared qualitative and quantitative changes in the lateral mobility of phospholipid molecules in the plasma membrane of intact cells under various conditions of specific interaction of integrins in the cell membrane with two extracellular matrix (ECM) components viz. fibronectin (FN) and laminin (LN). We found a strong and specific correlation between the lower lateral mobility of phosphatidylcholine (PC) and higher lateral mobility of phosphatidylethanolamine (PE) when cells were expressing high levels of alpha5beta1 integrin and thus were adherent and motile on FN. The interaction between PC and FN in alpha5 integrin expressing cells was aided by the strong affinity of alpha5 integrin to the FN matrix. Cholesterol was involved in regulating the lateral mobility of PC to a great extent and of PE to a lesser extent without affecting the overall microviscosity of the plasma membrane or the distribution of caveolin-marked domains. The distribution and mobility of PC and PE molecules in the lamellipodial regions differed from that in the rest of the membrane and also in the more motile and in the less motile cells. We propose that these differences in distribution of PC and PE in different regions of cell membrane and their respective lateral mobility are observed due to the specific interaction of PC molecules with FN molecules in the ECM. Our results outline a new role of integrin-matrix interactions in the regulation of membrane phospholipid behavior.     

Guinea pig fertilin exhibits restricted lateral mobility in epididymal sperm and becomes freely diffusing during capacitation

The guinea pig sperm protein fertilin functions in sperm-egg plasma membrane binding. Fertilin is initially present in the plasma membrane of the whole head in testicular sperm, then becomes concentrated into the posterior head domain during epididymal passage. Fertilin remains localized to the posterior head plasma membrane following the acrosome reaction, when it functions in sperm-egg interaction. Fluorescence redistribution after photobleaching was used to examine the lateral mobility of fertilin in both acrosome-intact and acrosome-reacted sperm. Fertilin exhibited highly restricted lateral mobility in both testicular and epididymal sperm (D < 10(-10) cm(2)/s). However, fertilin in acrosome-reacted sperm was highly mobile within the membrane bilayer (D = 1.8 x 10(-9) cm(2)/s and %R = 84). Measurement of the lateral mobility of fertilin in capacitated, acrosome-intact sperm revealed two populations of cells. In approximately one-half of the cells, lateral mobility of fertilin was similar to sperm freshly isolated from the cauda epididymis; while in the other half fertilin was highly mobile. The release of fertilin from interactions that restrict its lateral mobility may regulate its function in sperm-egg interaction.     

Electron transport between cytochrome c and alpha tocopherol.

Using liposomes we have demonstrated an electron transfer between tocopherol (vitamin E) and cytochrome c. Reduced cytochrome c protects vitamin E from oxidation induced either directly by ultraviolet light or indirectly by soybean lipoxygenase-catalyzed oxidation of arachidonic acid. Oxidized cytochrome c is reduced by tocopherol and tocopherol homologues (chromanols) resulting in accumulation of tocopheroxyl radicals which we detected by ESR. The peak height of the ESR spectrum of tocopheroxyl radicals (which is proportional to the amount of radical present) is proportional to the ratio of reduced to oxidized cytochrome c. In mitochondrial membranes succinate-cytochrome c reduction is inhibited by antimycin A. Addition of exogenous chromanols facilitates a by-pass of the antimycin A blocked electron pathway, and succinate-dependent cytochrome c reductase activity is restored. Cytochrome c may act as a water-soluble complement to the lipid-soluble ubiquinol in regenerating mitochondrial tocopherol from tocopheroxyl radical.     

Intracellular Ca2+ and not the extracellular matrix determines surface dynamics of AMPA-type glutamate receptors on aspiny neurons

The perisynaptic extracellular matrix (ECM) contributes to the control of the lateral mobility of AMPA-type glutamate receptors (AMPARs) at spine synapses of principal hippocampal neurons. Here, we have studied the effect of the ECM on the lateral mobility of AMPARs at shaft synapses of aspiny interneurons. Single particle tracking experiments revealed that the removal of the hyaluronan-based ECM with hyaluronidase does not affect lateral receptor mobility on the timescale of seconds. Similarly, cross-linking with specific antibodies against the extracellular domain of the GluA1 receptor subunit, which affects lateral receptor mobility on spiny neurons, does not influence receptor mobility on aspiny neurons. AMPARs on aspiny interneurons are characterized by strong inward rectification indicating a significant fraction of Ca²⁺-permeable receptors. Therefore, we tested whether Ca²⁺ controls AMPAR mobility in these neurons. Application of the membrane-permeable Ca²⁺ chelator BAPTA-AM significantly increased the lateral mobility of GluA1-containing synaptic and extrasynaptic receptors. These data indicate that the perisynaptic ECM affects the lateral mobility differently on spiny and aspiny neurons. Although ECM structures on interneurons appear much more prominent, their influence on AMPAR mobility seems to be negligible at short timescales.     

Increased rate of capping of concanavalin A receptors during early Xenopus development is related to changes in protein and lipid mobility

The mobility characteristics of plasma membrane constituents were studied in dissociated cells from embryos of Xenopus laevis at various stages of development from early blastula until neurulation. An increased rate of fluorescein isothiocyanate-concanavalin A induced patching and capping of Con A-binding proteins during this period of development was correlated with a threefold increase in the lateral mobility of the receptor molecules, as determined by the fluorescent photobleaching recovery (FPR) method, the major change occurring at the onset of gastrulation. Using the same method, it was demonstrated that the lateral mobility of plasma membrane lipids increases twofold during this period of development. The major change being detectable, however, at the late blastula stage. This is in coincidence with the initiation of cell motility in dissociated Xenopus embryo cells. It is concluded that the lateral mobility of membrane proteins and lipids increases significantly during early Xenopus development, but are at least in part subject to different control mechanisms. The results suggest that the initiation of morphogenetic movements is related to changes in the dynamic properties of plasma membrane constituents.     

Synthesis of β-Tubanol

Synthesis of β-tubanol (VIa) was achieved by the bromination of dihydro-β-tubanol acetate (VIIb) followed by dehydrobromination and the subsequent hydrolysis. 2,2-Dimethyl-5-hydroxy-chromanone as well as its derivatives (IV) and the corresponding chromanols (V) and chromans (VII) were also prepared.     

三种细胞系(人胚肺与小儿包皮成纤维及人胃癌细胞)膜表面ConA受体的再分布和侧向扩散的研究

利用荧光染色和荧光漂白后恢复FRAP技术观察人胚肺成纤维细胞,小儿包皮成纤维细胞和人胃腺癌细胞膜表面Con A受体复合物的分布及其侧向运动.结果表明:它们的分布呈均匀连续性,极为相似.但它们的侧向运动速率各异.两种成纤维细胞差异较小,而人胃癌细胞的侧向扩散系数显著地不同于两种成纤维细胞.因此把凝集素受体的分布与运动行为表达结合起来分析,能更好地认识细胞在生长、分化或恶变过程中膜性状的差异性与共性.     

The lateral mobility of cell membrane components is not altered following cell fusion induced by Sendai virus

The interaction of Sendai virus glycoproteins with cell membranes was proposed to increase the lateral mobility of membrane proteins, enabling membrane fusion and the aggregation of intramembrane particles by thermotropic separation (Volsky, DJ & Loyter, A, Biochim biophys acta 514 (1978) 213 [13]; Maeda, T et al. Exp cell res 123 (1979) 333 [15]; and Kim, J & Okada, Y, Exp cell res 132 (1981) 125 [44]). In order to test this hypothesis, we employed fluorescence photobleaching recovery to investigate the effects of Sendai virus-induced fusion on the lateral mobility of membrane proteins and lipids in a variety of cell types (human erythrocytes, BHK21, HeLa, 3T3 NIH, and mouse spleen lymphocytes). The results of the lateral diffusion measurements demonstrate that no significant alterations occur in the lateral motion of membrane proteins or a fluorescent phospholipid on all the cell types examined, including cells which revealed high susceptibility to the virally mediated fusion (human erythrocytes and BHK21 cells). These findings suggest that a permanent increase in the lateral mobility of cell surface components does not generally occur during Sendai virus-induced cell fusion, and thus cannot play a role in the fusion mechanism. The possible involvement of transient alterations in the lateral mobility of membrane components in the fusion mechanism is discussed.     

Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.     

Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.     

Lateral mobility of beta-receptors involved in adenylate cyclase activation

Cationized ferritin was found to inhibit the lateral mobility of intramembrane proteins in turkey erythrocyte membranes and the activation of adenylate cyclase by the (--)-epinephrine-bound beta-adrenergic receptor. It was observed that cationized ferritin has only a small direct effect on the beta-receptor and on the adenylate cyclase moiety. It is concluded that the cationized ferritin-induced inhibition of the hormone-dependent cyclase activity results from the inhibition of the lateral mobility of the receptor and therefore a decrease in the bimolecular rate of interaction between the receptor and the enzyme.     

On the decrease in lateral mobility of phospholipids by sugars

Upon cold and drought stress, sucrose and trehalose protect membrane structures from fusion and leakage. Similarly, these sugars protect membrane proteins from inactivation during dehydration. We studied the interactions between sugars and phospholipid membranes in giant unilamellar vesicles with the fluorescent lipid analog 3,3′-dioctadecyloxacarbocyanine perchlorate incorporated. Using fluorescence correlation spectroscopy, it was found that sucrose decreased the lateral mobility of phospholipids in the fully rehydrated, liquid crystalline membrane more than other sugars did, including trehalose. To describe the nature of the difference in the interaction of phospholipids with sucrose and trehalose, atomistic molecular dynamics studies were performed. Simulations up to 100 ns showed that sucrose interacted with more phospholipid headgroups simultaneously than trehalose, resulting in a larger decrease of the lateral mobility. Using coarse-grained molecular dynamics, we show that this increase in interactions can lead to a relatively large decrease in lateral phospholipid mobility.     

Restricted lateral mobility of plasma membrane CD4 impairs HIV-1 envelope glycoprotein mediated fusion

We investigated the effect of receptor mobility on HIV-1 envelope glycoprotein (Env)-triggered fusion using B16 mouse melanoma cells that are engineered to express CD4 and CXCR4 or CCR5. These engineered cells are resistant to fusion mediated CD4-dependent HIV-1 envelope glycoprotein. Receptor mobility was measured by fluorescence recovery after photobleaching (FRAP) using either fluorescently-labeled antibodies or transient expression of GFP-tagged receptors in the cells. No significant differences between B16 and NIH3T3 (fusion-permissive) cells were seen in lateral mobility of CCR5 or lipid probes. By contrast CD4 mobility in B16 cells was about seven-fold reduced compared to its mobility in fusion-permissive NIH3T3 cells. However, a CD4 mutant (RA5) that localizes to non-raft membrane microdomains exhibited a three-fold increased mobility in B16 cells as compared with WT-CD4. Interestingly, the B16 cells expressing the RA5 mutant (but not the wild type CD4) and coreceptors supported HIV-1 Env-mediated fusion. Our data demonstrate that the lateral mobility of CD4 is an important determinant of HIV-1 fusion/entry.     

The adenylate cyclase-coupled vasopressin V2-receptor is highly laterally mobile in membranes of LLC-PK1 renal epithelial cells at physiological temperature.

The lateral mobility of membrane-associated hormone receptors has been proposed to play an important role in signal transduction. Direct measurements, however, have shown that the receptors for insulin, epidermal growth factor and beta-adrenergic antagonists exhibit low mobility at physiological temperature. The present study, which represents the first report of lateral mobility of a polypeptide hormone receptor coupled to adenylate cyclase, yielded quite different results. The lateral mobility of the vasopressin renal-type (V2)-receptor was measured in the basal plasma membrane of cells of the LLC-PK1 porcine epithelial line, using the technique of fluorescence microphotolysis (photobleaching) and a rhodamine-labelled analogue of vasopressin. The analogue, 1-deamino[8-lysine(N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP) was synthesized and shown to have binding properties and biological activities very similar to those of Arg8-vasopressin (AVP). TR-LVP could be used to label specifically the V2-receptor of living LLC-PK1 cells, whereby LLC-PK1 cells incubated with TR-LVP in the presence of a 100-fold excess of AVP, or cells from the LLC-PK1 V2-receptor-deficient line M18 incubated with TR-LVP could be used as controls for non-specific binding. Using optical sectioning, specific receptor mobility could be measured both in the absence and presence of free TR-LVP. The V2-receptor was found to be largely mobile at 37 degrees C: the mobile fraction (f) was approximately 0.9, and the apparent lateral diffusion coefficient (D) approximately 3.0 X 10(-10) cm2/s. V2-receptor mobility greatly decreased with decreasing temperature: at 10 degrees C f was reduced to approximately 0.1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeastSaccharomyces cerevisiae

Summary We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (dil) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma mem branesof Saccharomyces cerevisiae inol andopi 3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobil ity was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe dil, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized sphero plasts, mobility was still somewhat lower than the mobility ob served in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.     

Lateral diffusion of phospholipids in the lipid surface of human low-density lipoprotein measured with a pyrenyl phospholipid probe

Human low-density lipoprotein (LDL) was labelled with the excimeric fluorescent phospholipid analogue 1-palmitoyl-2-(1'-pyreneoctanoyl)-sn-glycero-3-phosphocholine by using phosphatidylcholine-specific transfer protein for the probe insertion. The lateral diffusivity of the probe in the phospholipid/cholesterol surface monolayer of LDL was determined from the measured dependence of the pyrene monomer fluorescence yield on probe concentration. The data were analyzed by the milling-crowd model (J. Eisinger et al. (1986) Biophys. J. 49, 987-1001] to obtain the short-range lateral diffusivity of the probe. The lateral mobility of the probe in LDL was compared to that in model lipid systems, i.e. in protein-free LDL-like lipid particles and in small unilamellar vesicles, with a phospholipid/cholesterol composition characteristic of LDL. This analysis with the probability PE = 1 for excimer production between nearest-neighbour probes gives the lower limits for f, the frequency of translational lipid--lipid exchanges of the probe of 0.62 x 10(8), 0.19 x 10(8) and 0.19 x 10(8)s-1 in LDL, LDL-like lipid particles, and small unilamellar vesicles, respectively. The lower limits for the corresponding lateral diffusion constants are 16, 5 and 5 microns 2 s-1. The results suggest that the translational mobility of phospholipid molecules in the lipid--protein surface of LDL is not constrained by the apolipoprotein B-100 moiety or the neutral lipid core of the lipoprotein. Instead, the protein moiety may perturb the lipid order with the lipid--associating peptide domains and thus fluidize the amphiphilic surface monolayer of LDL relative to the protein-free model systems. In general, lateral diffusivity of the pyrenyl phospholipid probe in LDL and the model lipid systems is comparable to the lateral mobility of lipid analogue probes in a variety of model and biological membranes.     

Clonal selection by fluorescence redistribution after photobleaching (FRAP)--a "fast" lateral mobility fibroblast mutant (E7G1)

Fluorescence redistribution after photobleaching (FRAP) was utilized to select a "fast" lateral mobility clone from Kirsten murine sarcoma virus-transformed 3T3 (KMSV-3T3) fibroblasts. The clone, E7G1, demonstrated a lateral mobility for membrane wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors of (2.1 +/- 1.6) X 10(-9) cm2/s and (2.7 +/- 2.3) X 10(-9) cm2/s, respectively. These mobilities were approximately equivalent to phospholipid mobility (2.8 +/- 1.9 X 10(-9) cm2/s). The fast mobility phenotype is observed when the cells are unattached and spherical. Upon attachment, the mobility decreases to (0.19 +/- 0.19) X 10(-9) cm2/s. In addition, the ability of Con A to initiate global modulation was completely lost in spread as well as spherical cells in the E7G1 fast mobility clone. A comparison of F-actin patterns between untransformed Balb/c fibroblasts and the E7G1-transformed line suggests a correlation between well-developed stress fiber assemblies and the ability to induce global modulation. The fast mobility clone was stable for at least 23 passages.     

Association of spectrin with its membrane attachment site restricts lateral mobility of human erythrocyte integral membrane proteins

Interactions between spectrin and the inner surface of the human erythrocyte membrane have been implicated in the control of lateral mobility of the integral membrane proteins. We report here that incubation of “leaky” erythrocytes with a water-soluble proteolytic fragment containing the membrane attachment site for spectrin achieves a selective and controlled dissociation of spectrin from the membrane, and increases the rate of lateral mobility of fluorescein isothiocyanate-labeled integral membrane proteins (> 70% of label in band 3 and PAS-1). Mobility of membrane proteins is measured as an increase in the percentage of uniformly fluorescent cells with time after fusion of fluorescent with nonfluorescent erythrocytes by Sendai virus. The cells are permeable to macromolecules since virus-fused erythrocytes lose most of their hemoglobin. The membrane attachment site for spectrin has been solubilized by limited proteolysis of inside-out erythrocyte vesicles and has been purified (V). Bennett, J Biol Chem 253:2292 (1978). This 72,000-dalton fragment binds to spectrin in solution, competitively inhibits association of ³²P-spectrin with inside-out vesicles with a K_i of 10^?7M, and causes rapid dissociation of ³²P-spectrin from vesicles. Both acid-treated 72,000-dalton fragment and the 45,000 dalton-cytoplasmic portion of band 3, which also was isolated from the proteolytic digest, have no effect on spectrin binding, release, or membrane protein mobility. The enhancement of membrane protein lateral mobility by the same polypeptide that inhibits binding of spectrin to inverted vesicles and displaces spectrin from these vesicles provides direct evidence that the interaction of spectrin with protein components in the membrane restricts the lateral mobility of integral membrane proteins in the erythrocyte.