Membrane effects of cocoa procyanidins in liposomes and Jurkat T cells

We investigated the effects of the interaction between flavanols and related procyanidins (dimer to hexamer) with both cell and synthetic membranes, on bilayer fluidity and susceptibility to oxidation. Cocoa derived dimers (0.05 to 1 microg/ml) protected Jurkat T cells from AMVN-mediated oxidation and increased plasma membrane fluidity. These effects occurred in a concentration- and chain length-dependent manner. In liposomes, procyanidins prevented the Fe2+ -induced permeabilization of the membrane. Together, these results support the hypothesis that procyanidins could interact with the polar headgroup of lipids, increasing membrane fluidity and also, preventing the access of molecules that could affect membrane integrity.     

The Interaction of Flavonoids with Membranes: Potential Determinant of Flavonoid Antioxidant Effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2′-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2′-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid–water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

The interaction of flavonoids with membranes: potential determinant of flavonoid antioxidant effects

Twenty six phenolic substances including representatives of the families, flavanones, flavanols and procyanidins, flavonols, isoflavones, phenolic acids and phenylpropanones were investigated for their effects on lipid oxidation, membrane fluidity and membrane integrity. The incubation of synthetic phosphatidylcholine (PC) liposomes in the presence of these phenolics caused the following effects: (a) flavanols, their related procyanidins and flavonols were the most active preventing 2,2'-azo-bis (2,4-dimethylvaleronitrile) (AMVN)-induced 2-thiobarituric acid-reactive substances (TBARS) formation, inducing lipid ordering at the water-lipid interface, and preventing Triton X-100-induced membrane disruption; (b) all the studied compounds inhibited lipid oxidation induced by the water-soluble oxidant 2,2'-azo-bis (2-amidinopropane) (AAPH), and no family-related effects were observed. The protective effects of the studied phenolics on membranes were mainly associated to the hydrophilicity of the compounds, the degree of flavanol oligomerization, and the number of hydroxyl groups in the molecule. The present results support the hypothesis that the chemical structure of phenolics conditions their interactions with membranes. The interactions of flavonoids with the polar head groups of phospholipids, at the lipid-water interface of membranes, should be considered among the factors that contribute to their antioxidant effects.     

Influence of oligomer chain length on the antioxidant activity of procyanidins

The antioxidant activity of catechin monomers and procyanidin (dimers to hexamers) fractions purified from cocoa was studied in two in vitro systems: liposomes and human LDL. Liposome oxidation (evaluated as formation of 2-thiobarbituric acid reactive substances) was initiated with 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), 2,2'-azobis (2,4-dimethylvaleronitrile) (AMVN), iron/ascorbate, or UV-C; LDL oxidation (evaluated as formation of conjugated dienes) was initiated with Cu(2+) or AAPH. Catechin monomers and procyanidin fractions inhibited both liposome and LDL oxidation. Monomers, dimers, and trimers fractions were the most effective antioxidants when liposome oxidation was initiated in the aqueous phase. When oxidation was initiated in the lipid domains, higher molecular weight procyanidins were the most effective. All fractions significantly inhibited Cu-mediated LDL oxidation; no significant effect of procyanidin molecular weight was observed. The hexamer fraction was the least effective with respect to preventing AAPH initiated LDL oxidation. Results reported herein give further evidence on the influence of the oligomer chain length on the antioxidant protection by procyanidins.     

Procyanidins can interact with Caco-2 cell membrane lipid rafts: Involvement of cholesterol

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-β-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol–Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex–cholesterol bondings. Procyanidin–lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.     

Inhibition of angiotensin converting enzyme (ACE) activity by flavan-3-ols and procyanidins

It was determined that flavan-3-ols and procyanidins have an inhibitory effect on angiotensin I converting enzyme (ACE) activity, and the effect was dependent on the number of epicatechin units forming the procyanidin. The inhibition by flavan-3-ols and procyanidins was competitive with the two substrates assayed: N-hippuryl-L-histidyl-L-leucine (HHL) and N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPGG). Tetramer and hexamer fractions were the more potent inhibitors, showing Ki of 5.6 and 4.7 microM, respectively. As ACE is a membrane protein, the interaction of flavanols and procyanidins with the enzyme could be related to the number of hydroxyl groups on the procyanidins, which determine their capacity to be adsorbed on the membrane surface.     

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.     

Cocoa procyanidin chain length does not determine ability to protect LDL from oxidation when monomer units are controlled

Cocoa flavan-3-ols (catechin, epicatechin and oligomeric procyanidins) were tested for their ability to decrease LDL oxidative susceptibility and spare alpha-tocopherol (alpha-toc) in vitro. Physiologic concentration (0.10-0.50 &mgr;M) of flavanols were used. The flavanols increased LDL conjugated diene lag times dose-dependently from 23-207% and 15-143% in response to copper and AAPH oxidation, respectively, and delayed alpha-toc consumption. Sparing of LDL alpha-toc represents a possible mechanism for flavanols to enhance the resistance of plasma and LDL to oxidative stress. Procyanidins decreased LDL oxidative susceptibility with increasing chain length. However, when based on equivalent amounts of monomeric units, they inhibited LDL oxidation to a similar extent. This suggests that antioxidant activity of procyanidins with biologic substrates is not attributable to chain length or charge delocalization through polymeric linkages, but primarily to ring structures and catechol groups. Additionally, human plasma was analyzed for the presence of oligomeric procyanidins following consumption of a flavanol-rich cocoa product. Procyanidin dimers were detected in plasma concordant with the appearance of monomeric flavanols, with a peak of 0.08 +/- 0.01 &mgr;mol/L (n = 6) at two hours after consumption. Thus, this paper confirms the occurrence of procyanidins in human plasma, and extends previous structure-function observations regarding flavanoid protection of LDL.     

Dietary flavonoids: Role of (-)-epicatechin and related procyanidins in cell signaling

Plant polyphenols are among the most abundant phytochemicals present in human diets. Increasing evidence supports the health-promoting effects of certain polyphenols, including flavonoids. This review discusses current knowledge of the capacity of monomeric flavanols, i.e., (−)-epicatechin and (+)-catechin, and their derived procyanidins to modulate cell signaling and the associations of these actions with better health. Flavanols and procyanidins can regulate cell signaling through different mechanisms of action. Monomers and dimeric procyanidins can be transported inside cells and directly interact and modulate the activity of signaling proteins and/or prevent oxidation. Larger and nonabsorbable procyanidins can regulate cell signaling by interacting with cell membrane proteins and lipids, inducing changes in membrane biophysics, and by modulating oxidant production. All these actions would be limited by the bioavailability of flavanols at the target tissue. The protection from cardiac and vascular disease and from cancer that is associated with a high consumption of fruit and vegetables could be in part explained by the capacity of flavanols and related procyanidins to modulate proinflammatory and oncogenic signals.     

Liposomes alter thermal phase behavior and composition of red blood cell membranes

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.     

Vitamin E: inhibition of retinol-induced hemolysis and membrane-stabilizing behavior.

For the elucidation of the mechanism of membrane stabilization by vitamin E, the effects of alpha-tocopherol and its model compounds on either retinol-induced hemolysis of rabbit erythrocytes or the permeability and fluidity of liposomal membranes have been studied. Retinol-induced rabbit erythrocyte hemolysis has been found not to be caused by the oxidative disruption of erythrocyte membrane lipids initiated by retinol oxidation, but rather to arise from physical damage of the membrane micelle induced by penetration of retinol molecules. In suppressing hemolysis, alpha-tocopherol was more effective than other naturally occurring tocopherols. alpha-Tocopheryl acetate, nicotinate, and 6-deoxy-alpha-tocopherol were more effective than alpha-tocopherol itself. The inhibitory effects of alpha-tocopherol model compounds having side chains with at least two isoprene units or a long straight chain instead of the isoprenoid side chain were similar to those of alpha-tocopherol. These data suggest that for protection of membranes against retinol-induced damage, the hydroxyl group of alpha-tocopherol is not critical, but rather the chroman ring, three methyl groups on the aromatic ring, and the long side chain are necessary. To verify the mechanism of the inhibitory effect on hemolysis, not only the effect of vitamin E and its model compounds on the membrane permeability and fluidity, but also the mobility of alpha-tocopherol molecule in membranes has been investigated using bilayer liposomes as the model membranes. Addition of alpha-tocopherol to membranes produced a greater decrease in the permeability and fluidity of rat liver phosphatidylcholine liposomes compared with egg yolk phosphatidylcholine liposomes. In dipalmitoylphosphatidylcholine liposomes, however, alpha-tocopherol was less effective, that is, the more unsaturated the lipids, the more they interact with alpha-tocopherol. 2,2,5,7,8-Pentamethyl-6-chromanol with no isoprenoid side chain and phytol without the chromanol moiety had no effect. The measurement of 13C NMR relaxation times revealed that the mobility of methyl groups on the aromatic ring of alpha-tocopherol in membranes is significantly restricted. In contrast, the methyl groups at positions 4'a and 8'a on the isoprenoid side chain have high degrees of motional freedom in the lipid core of membranes. Furthermore, it was found that alpha-tocopherol in membranes interacts with chromate ions added as potassium chromate outside the membranes, resulting in an increase in membrane fluidity. These results are compatible with those of the inhibitory effect on retinol-induced erythrocyte hemolysis. On the basis of the results obtained here, a possible mechanism for membrane stabilization by vitamin E is proposed.     

Inhibitory effects of cocoa flavanols and procyanidin oligomers on free radical-induced erythrocyte hemolysis

Excessive peroxidation of biomembranes is thought to contribute to the initiation and progression of numerous degenerative diseases. The present study examined the inhibitory effects of a cocoa extract, individual cocoa flavanols (-)-epicatechin and (+)-catechin, and procyanidin oligomers (dimer to decamer) isolated from cocoa on rat erythrocyte hemolysis. In vitro, the flavanols and the procyanidin oligomers exhibited dose-dependent protection against 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis between concentrations of 2.5 and 40 microM. Dimer, trimer, and tetramer showed the strongest inhibitory effects at 10 microM, 59.4%, 66.2%, 70.9%; 20 microM, 84.1%, 87.6%, 81.0%; and 40 microM, 90.2%, 88.9%, 78.6%, respectively. In a subsequent experiment, male Sprague-Dawley rats (approximately 200 g; n = 5-6) were given a 100-mg intragastric dose of a cocoa extract. Blood was collected over a 4-hr time period. Epicatechin and catechin, and the dimers (-)-epicatechin-(4beta>8)-epicatechin (Dimer B2) and (-)-epicatechin-(4beta>6)-epicatechin (Dimer B5) were detected in the plasma with concentrations of 6.4 microM, and 217.6, 248.2, and 55.4 nM, respectively. Plasma antioxidant capacity (as measured by the total antioxidant potential [TRAP] assay) was elevated (P < 0.05) between 30 and 240 min following the cocoa extract feeding. Erythrocytes obtained from the cocoa extract-fed animals showed an enhanced resistance to hemolysis (P < 0.05). This enhanced resistance was also observed when erythrocytes from animals fed the cocoa extract were mixed with plasma obtained from animals given water only. Conversely, plasma obtained from rats given the cocoa extract improved the resistance of erythrocytes obtained from rats given water only. These results show cocoa flavanols and procyanidins can provide membrane protective effects.     

Stability of heterochiral hybrid membrane made of bacterial sn-G3P lipids and archaeal sn-G1P lipids

The structure of membrane lipids in Archaea is different from those of Bacteria and Eucarya in many ways including the chirality of the glycerol backbone. Until now, heterochiral membranes were believed to be unstable; thus, no cellular organism could have existed before the separation of the groups of life. In this study, we tested the formation of heterochiral hybrid membrane made of Bacterial sn-glycerol-3-phosphate-type polar lipid and Archaeal sn-glycerol-1-phosphate-type polar lipid using the fluorescence probe. The stability of the hybrid liposomes made of phosphatidylethanolamines or phosphatidylcholines or polar lipids of thermophilic Bacteria and polar lipids of Archaea were investigated. The hybrid liposomes are all stable compared with homochiral liposome made of dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine. However, the stability was drastically changed with increasing carbon chain length. Accordingly, "chirality" may not be but chain length is important. From these results, we suggest that the heterochiral hybrid membrane could be used as the membrane lipid for the last universal common ancestor (Commonote) before the emergence of Archaea and Bacteria.     

Liposomes as model for taste cells: receptor sites for bitter substances including N-C=S substances and mechanism of membrane potential changes

Various bitter substances were found to depolarize liposomes. The results obtained are as follows: (1) Changes in the membrane potential of azolectin liposomes in response to various bitter substances were monitored by measuring changes in the fluorescence intensity of 3,3'-dipropylthiocarbocyanine iodide [diS-C3(5)]. All the bitter substances examined increased the fluorescence intensity of the liposome-dye suspension, which indicates that the substances depolarize the liposomes. There existed a good correlation between the minimum concentrations of the bitter substances to depolarize the liposomes and the taste thresholds in humans. (2) The effects of changed lipid composition of liposomes on the responses to various bitter substances vary greatly among bitter substances, suggesting that the receptor sites for bitter substances are multiple. The responses to N-C=S substances and sucrose octaacetate especially greatly depended on the lipid composition; these compounds depolarized only liposomes having certain lipid composition, while no or hyperpolarizing responses to these compounds were observed in other liposomes examined. This suggested that the difference in "taster" and "nontaster" for these substances can be explained in terms of difference in the lipid composition of taste receptor membranes. (3) It was confirmed that the membrane potential of the planar lipid bilayer is changed in response to bitter substances. The membrane potential changes in the planar lipid bilayer as well as in liposomes in response to the bitter substances occurred under the condition that there is no ion gradient across the membranes. These results suggested that the membrane potential changes in response to bitter substances stem from the phase boundary potential changes induced by adsorption of the substances on the hydrophobic region of the membranes.     

Membrane potential-dependent binding of polysialic acid to lipid monolayers and bilayers

Polysialic acids are linear polysaccharides composed of sialic acid monomers. These polyanionic chains are usually membrane-bound, and are expressed on the surfaces of neural, tumor and neuroinvasive bacterial cells. We used toluidine blue spectroscopy, the Langmuir monolayer technique and fluorescence spectroscopy to study the effects of membrane surface potential and transmembrane potential on the binding of polysialic acids to lipid bilayers and monolayers. Polysialic acid free in solution was added to the bathing solution to assess the metachromatic shift in the absorption spectra of toluidine blue, the temperature dependence of the fluorescence anisotropy of DPH in liposomes, the limiting molecular area in lipid monolayers, and the fluorescence spectroscopy of oxonol V in liposomes. Our results show that both a positive surface potential and a positive transmembrane potential inside the vesicles can facilitate the binding of polysialic acid chains to model lipid membranes. These observations suggest that these membrane potentials can also affect the polysialic acid-mediated interaction between cells.     

Localization of the active center of microsomal cytochrome P-450

To solve the problem of localization of the active center of cytochrome P-450 in microsomal membranes, new bifunctional compounds (I-IV), which contain pyridine radical, aliphatic chain of variable length and diphosphonic acid ("floating" molecules) have been applied. These compounds inhibit oxidation and binding of the substrates of cytochrome P-450 (aminopyrine and aniline), inhibition being of a competitive character. Measurements of distribution coefficients between water and membranes of microsomes and liposomes from egg phosphatidylcholine evidence that the microsomal proteins are necessary for providing effective interaction of I-IV with microsomal membrane. The 1H-NMR method has demonstrated compounds to be incorporated into lipid bilayer so that the non-polar part is in the inner membrane volume. The results obtained confirm our previous conclusion (Krainev A.G., Weiner L.M., Alferyev I.S., Slynko N.M. (1985) Biochim. Biophys. Acta, 818, 96-104) about localization of the active center of microsomal cytochrome P-450 at the depth of approximately 18 A from the hydrophilic surface of a membrane.     

Biological activity of new N-oxides of tertiary amines

Potential biological properties of newly synthesized single and double alkyl chain N-oxides of tertiary amines (NTA) were studied. Individual compounds in each of the series had alkyl chains of different length. Various experiments were performed to determine a mechanism of the interaction between NTA and model and biological membranes. These were measurements of hemolytic efficiencies of NTA (pig erythrocytes), their influence on the transition temperatures (DPPC liposomes), on potassium leakage from cucumber, its growth and chlorophyll content (Cucumis sativus cv. Krak F1), and on the resting membrane potential in alga cells (Nitellopsis obtusa). Also, prevention of erythrocyte membrane lipid oxidation induced by UV irradiation was studied. Potential antioxidative properties of NTA were additionally tested in radical chromogen (ABTS) experiments in which antioxidative efficiencies of NTA were compared to that of the standard antioxidant Trolox. It was found that NTA readily interacted with erythrocyte membranes. Their hemolyzing efficiency increased with the alkyl chain length. Slightly more intensive interaction was found for double alkyl chain compounds. Similar results were obtained in DSC experiments, where incorporation of NTA into liposomal membranes shifted the main transition temperatures and caused a broadening of the main transition peaks depending on the alkyl chain length. Double alkyl chain compounds were also found more efficiently influencing the growth of cucumber. Influence of NTA on the resting membrane potential of algae cells was not quite following the alkyl chain length rule found in erythrocyte and liposome experiments. Also potassium leakage and chlorophyll content determined in physiological experiments were not following the increase of lipophilicity of compounds. Most efficiently influencing those parameters were NTA having shorter alkyl chains, and efficiencies of single alkyl chain compounds were evidently stronger. Both methods used to test the antioxidative properties of NTA showed that they depended on the alkyl chain lengths of compounds within each series, but double alkyl chain ones exhibited markedly greater efficiency.     

Relevance of lipid polar headgroups on boron-mediated changes in membrane physical properties

Using liposomes composed of either brain phosphatidylcholine (PC), or binary mixtures of PC and phosphatidylserine (PS), galactolipids (GL), phosphatidylinositol (PI), cardiolipin (CL), phosphatidic acid (PA), or phosphatidylethanolamine (PE), we investigated the effects of graded amounts of boric acid (B, 0.5-1000 microM) on the following membrane physical properties: (a) surface potential, (b) lipid rearrangement through lateral phase separation, (c) fluidity, and (d) hydration. Incubation of the different populations of vesicles with B was associated with a small, but statistically significant, increase in membrane surface potential in PC, PC:PS, PC:GL, PC:PI, PC:PA, and PC:PE liposomes. B-induced lipid lateral rearrangement through lateral phase separation in PC, PC:PA, and PC:PE liposomes; but had no effects on PC:PS, PC:GL, and PC:PI liposomes. In PC liposomes B affected membrane fluidity at the water-lipid interface without affecting the hydrophobic core of the bilayer. In all the other binary liposomes studied, B increased membrane fluidity in both, the hydrophobic portion of the membrane and in the anionic domains. The above was associated with a decrease in the fluidity of the cationic domains. B (10-1000 microM) decreased membrane hydration regardless the composition of the liposomes. The obtained results demonstrate the ability of B to interact with membranes, and induce changes in membrane physical properties. Importantly, the extent of B-membrane interactions and the consequent effects were dependent on the nature of the lipid molecule; as such, B had greater affinity with lipids containing polyhydroxylated moieties such as GL and PI. These differential interactions may result in different B-induced modulations of membrane-associated processes in cells.     

The antioxidant properties of zinc: interactions with iron and antioxidants.

Potential mechanisms underlying zinc's capacity to protect membranes from lipid oxidation were examined in liposomes. Using lipid oxidation initiators with different chemical and physical properties (transition metals, lipid- or water-soluble azo compounds, ultraviolet radiation c (UVc), superoxide radical anion (O2*-), and peroxynitrite (ONOO-) we observed that zinc only prevented copper (Cu2+)- and iron (Fe2+)-initiated lipid oxidation. In the presence of Fe2+, the antioxidant action of zinc depended directly on the negative charge density of the membrane bilayer. An inverse correlation (r2: 0.96) was observed between the capacity of zinc to prevent iron binding to the membrane and the inhibitory effect of zinc on Fe2+-initiated lipid oxidation. The interaction of zinc with the bilayer did not affect physical properties of the membrane, including rigidification and lateral phase separation known to increase lipid oxidation rates. The interactions between zinc and the lipid- (alpha-tocopherol) and water- (epicatechin) soluble antioxidants were studied. The inhibition of Fe2+-induced lipid oxidation by either alpha-tocopherol or epicatechin was increased by the simultaneous addition of zinc. The combined actions of alpha-tocopherol (0.01 mol%), epicatechin (0.5 microM) and zinc (5-50 microM) almost completely prevented Fe2+ (25 microM)-initiated lipid oxidation. These results show that zinc can protect membranes from iron-initiated lipid oxidation by occupying negatively charged sites with potential iron binding capacity. In addition, the synergistic actions of zinc with lipid and water-soluble antioxidants to prevent lipid oxidation, suggests that zinc is a pivotal component of the antioxidant defense network that protects membranes from oxidation.     

Lateral diffusion of ubiquinone during electron transfer in phospholipid- and ubiquinone-enriched mitochondrial membranes

After fusion of small unilamellar phospholipid liposomes with mitochondrial inner membranes, the rate of electron transfer between membrane dehydrogenases and cytochrome c decreases as the average distance between integral membrane proteins increases, suggesting that electron transfer is mediated through a diffusional process in the membrane plane (Schneider, H., Lemasters, J. J., H?chli, M., and Hackenbrock, C. R. (1980)., J. Biol. Chem. 255, 3748-3756). The role of ubiquinone in this process was evaluated by fusing liposomes containing ubiquinone-10 or ubiquinone-6, with inner membranes. In control membranes enriched with phospholipid only, ubiquinol-cytochrome c reductase and NADH- and succinate-cytochrome c reductase activities decreased proportionally to the increase in bilayer lipid. These decreases were restored substantially in phospholipid plus ubiquinone-supplemented membranes. The degree to which restoration occurred was dependent upon the length of the isoprenoid side chain of the ubiquinone with the shorter chain length ubiquinone-6, always giving greater restoration than ubiquinone-10. It is concluded that electron transfer between flavin-linked dehydrogenases (Complexes I and II) and cytochrome bc1 (Complex III) occurs by independent, lateral diffusion of ubiquinone as well as independent, lateral diffusion of ubiquinone as well as the protein complexes within the plane of the membrane.