Permeation of a beta-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled beta-heptapeptide fluoresceinyl-NH-CS-(S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the beta-heptapeptide (S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb(3+)-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled beta-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular beta-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.     

Supported phospholipid bilayers.

Phospholipid bilayers have been formed on glass, quartz, and silicon surfaces by a sequential transfer of two monolayers at a pressure of approximately 40 dyn/cm from the air-water interface to the solid substrates. Lateral diffusion measurements of L-alpha-dipalmitoylphosphatidylcholine (DPPC) bilayers supported on oxidized silicon wafers reveal two sharp phase transitions at temperatures similar to those found in multilayer systems with several different techniques. The diffusion measurements obtained using fluorescence recovery after pattern photobleaching provide evidence for the existence of an intermediate (probably P beta' or ripple) phase in single bilayers. While in the intermediate and high temperature (liquid-crystalline L alpha) phase, the diffusion coefficients do not vary very much with temperature, a strong temperature dependence is observed in the low temperature (gel L beta') phase. This is attributed to defect-mediated diffusion. Lipids in silicon supported bilayers made from L-alpha-dioleoylphosphatidylcholine (DOPC) or L-alpha-dimyristoylphosphatidylcholine (DMPC) diffuse rapidly above their respective chain-melting transition temperatures. Arrhenius plots show straight lines with activation energies of 40.9 and 43.7 kJ/mol, respectively. Supported DPPC bilayers on oxidized silicon form long tubular liposomes when heated through their oxidized silicon form long tubular liposomes when heated through their chain-melting-phase transition, as viewed with epifluorescence microscopy. It is suggested that this is a consequence of the expansion of the lipid on the fixed solid support. Conversely, DOPC bilayers form large void areas on this substrate upon cooling. Large circular membrane defects (holes) are observed under rapid coating conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Permeation of protons, potassium ions, and small polar molecules through phospholipid bilayers as a function of membrane thickness.

Two mechanisms have been proposed to account for solute permeation of lipid bilayers. Partitioning into the hydrophobic phase of the bilayer, followed by diffusion, is accepted by many for the permeation of water and other small neutral solutes, but transient pores have also been proposed to account for both water and ionic solute permeation. These two mechanisms make distinctively different predictions about the permeability coefficient as a function of bilayer thickness. Whereas the solubility-diffusion mechanism predicts only a modest variation related to bilayer thickness, the pore model predicts an exponential relationship. To test these models, we measured the permeability of phospholipid bilayers to protons, potassium ions, water, urea, and glycerol. Bilayers were prepared as liposomes, and thickness was varied systematically by using unsaturated lipids with chain lengths ranging from 14 to 24 carbon atoms. The permeability coefficient of water and neutral polar solutes displayed a modest dependence on bilayer thickness, with an approximately linear fivefold decrease as the carbon number varied from 14 to 24 atoms. In contrast, the permeability to protons and potassium ions decreased sharply by two orders of magnitude between 14 and 18 carbon atoms, and leveled off, when the chain length was further extended to 24 carbon atoms. The results for water and the neutral permeating solutes are best explained by the solubility-diffusion mechanism. The results for protons and potassium ions in shorter-chain lipids are consistent with the transient pore model, but better fit the theoretical line predicted by the solubility-diffusion model at longer chain lengths.     

Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayers.

Dimethyl sulfoxide (Me2SO) is a widely used cryoprotectant for biological structures such as membranes. Despite hundreds of studies on the effects of this molecule, surprisingly little is known about its cryoprotective mechanism. This study investigates the ability of various Me2SO analogs to serve as cryoprotectants for liposomes. The data show that an increase in hydrophobicity progressively reduces the cryoprotective effect of sulfoxides. Additional experiments using phospholipid vesicles of varying composition demonstrate the Me2SO is markedly less effective on liposomes carrying a net negative charge. In fact, cryoprotection by Me2SO was virtually eliminated in vesicles composed of 30% phosphatidylserine (a negatively charged lipid). Based on these results, we suggest that the polar sulfoxide moiety of Me2SO interacts electrostatically with phospholipid membranes and that this interaction is critical for Me2SO's cryoprotective effect for membranes.     

Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) is known to protect isolated enzymes during freezing while destabilizing proteins at high temperatures. This apparent paradox is the subject of a review by Arakawa et al. ((1990) Cryobiology 27, 401-415), who present evidence for a temperature-dependent, hydrophobic interaction between DMSO and non-polar moieties of proteins. The present study investigates the interaction of DMSO with phospholipid bilayers. Phospholipid vesicles containing carboxyfluorescein were exposed to several concentrations of DMSO at various temperatures. Leakage rates increased with DMSO concentration and temperature. This effect was not reduced in the presence of solutes that have been shown to neutralize DMSO toxicity in tissues. The increased leakage rates correlate well with the increased partitioning of DMSO from water to octanol at higher temperatures. Additionally, reductions in the CH2 vibrations of the bilayer are also shown to depend on DMSO concentration and temperature. A similar reduction in CH2 vibrations was observed in solutions of octanol and DMSO, suggesting that this effect is not mediated through an interaction with water. Furthermore, investigation of sulfoxide vibrations indicate that DMSO is not hydrogen bonded to the alcohol moiety of octanol, and therefore the interaction between DMSO and octanol is most likely due to a hydrophobic association. These results are consistent with a destabilization of phospholipid membranes at higher temperatures due to a hydrophobic association between DMSO and the bilayer.     

Permeation of phospholipid membranes by peroxynitrite

Quantitative kinetic models have been developed for the reaction between peroxynitrite and membrane lipids in vesicles and for transmembrane oxidation of reactants located within their inner aqueous cores. The models were used to analyze TBARS formation and oxidation of entrapped Fe(CN)(6)(4)(-) ion in egg lecithin liposomes and several artificial vesicles. The analyses indicate that permeation of the bilayers by ONOOH and NO(2)(*), a radical formed by homolysis of the ONOOH bond, is unusually rapid but that permeation by ONOO(-) and CO(3)(*)(-), a radical formed when CO(2) is present, is negligible. Bicarbonate protects the vesicles against both membrane and Fe(CN)(6)(4)(-) oxidation by rapid competitive CO(2)-catalyzed isomerization of ONOOH to NO(3)(-); this effect is partially reversed by addition of nitrite ion, which reacts with CO(3)(*)(-) to generate additional NO(2)(*). Under medium conditions mimicking the physiological milieu, a significant fraction of the oxidants escape to inflict damage upon the vesicular assemblies. Rate constants for several elementary reaction steps, including transmembrane diffusion rates for ONOOH and NO(2)(*), were estimated from the bicarbonate dependence of the oxidative reactions.     

Perturbation of phospholipid bilayers by DDT

The localization of the effects of DDT (5–50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23°C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54°C or egg PC at 23°C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.     

The monensin-mediated transport of sodium ions through phospholipid bilayers studied by 23Na-NMR spectroscopy

The monensin-mediated transport of sodium ions through the walls of large unilamellar vesicles of egg phosphatidylcholine was studied using 23Na-NMR and aqueous shift reagents. The transport is dynamic on the NMR time-scale and is strictly first order in monensin over the concentration ranges studied indicating that transport occurs by a 1:1 Na+-ionophore complex. Transport appears to be inhibited by increasing concentrations of Na+.     

Modulated phases of phospholipid bilayers induced by tocopherols

The influence of α-, γ- and δ-tocopherols on the structure and phase behavior of dipalmitoyl phosphatidylcholine (DPPC) bilayers has been determined from X-ray diffraction studies on oriented multilayers. In all the three cases the main-transition temperature (T(m)) of DPPC was found to decrease with increasing tocopherol concentration up to around 25 mol%. Beyond this the main transition is suppressed in the case of γ-tocopherol, whereas T(m) becomes insensitive to composition in the other two cases. The pre-transition is found to be suppressed over a narrow tocopherol concentration range between 7.5 and 10 mol% in DPPC-γ-tocopherol and DPPC-δ-tocopherol bilayers, and the ripple phase occurs down to the lowest temperature studied. In all the three cases a modulated phase is observed above a tocopherol concentration of about 10 mol%, which is similar to the P(β) phase reported in DPPC-cholesterol bilayers. This phase is found to occur even in excess water conditions at lower tocopherol concentrations, and consists of bilayers with periodic height modulation. These results indicate the ability of tocopherols to induce local curvature in membranes, which could be important for some of their biological functions.     

Micromanipulation of phospholipid bilayers by atomic force microscopy

The molecular details of adhesion mechanics in phospholipid bilayers have been studied using atomic force microscopy (AFM). Under tension fused bilayers of dipalmitoylphosphatidylcholine (DPPC) yield to give non-distance dependent and discrete force plateaux of 45.4, 81.6 and 113+/-3.5 pN. This behaviour may persist over distances as great as 400 nm and suggests the stable formation of a cylindrical tube which bridges the bilayers on the two surfaces. The stability of this connective structure may have implications for the formation of pili and hence for the initial stage of bacterial conjugation. Dimyristoylphosphatidylcholine (DMPC) bilayers also exhibit force plateaux but with a much less pronounced quantization. Bilayers composed of egg PC, sterylamine and cholesterol stressed in a similar way show complex behaviour which can in part be explained using the models demonstrated in the pure lipids.     

On the mechanism of non-electrolyte permeation through lipid bilayers and through biomembranes

The temperature effects on the permeation of polyhydroxy alcohols through the lipid bilayers of liposomes with a great variety in chemical composition were studied. Although important differences in the permeability of the various lipid bilayers were observed, Arrhenius plots demonstrated that the activation energy is independent of the degree of unsaturation or the presence of cholesterol in the paraffin barriers. The activation energies found for the penetration of a bilayer with a liquid paraffin core are 14.3 kcal for glycol, 19.4 kcal for glycerol, and 20.8 kcal for erythritol. These values are in agreement with the energies that can be expected for complete dehydration of the permeant molecules. The idea that the activation energy is determined by the number of hydrogen bonds with water is supported by the finding that a series of different diols did demonstrate practically identical activation energies. Studies on a number of biological membranes demonstrated the same activation energies for the penetration of glycerol and erythritol as found in the experiments with liposomes. These facts support the view that both the lipid bilayers and the biological membranes are penetrated by single fully dehydrated molecules.     

Adenylyl cyclase activation by halide anions other than fluoride

Adenylyl cyclase of rat liver and fat cells is activated by chloride, bromide, and iodide in addition to fluoride, previously believed to be uniquely effective among the halide anions. Liver homogenates are activated approximately 6 fold by fluoride while chloride and bromide increase cyclase by 3 fold and iodide about 2 fold. Optimal concentrations of chloride, bromide and iodide are about 100 times higher than those required for activation by fluoride. The cyclase of fat cell ghosts is activated some 9 fold by fluoride, but the other halide anions produced effects very similar in magnitude to those seen with liver, although for fat the optimally effective concentrations were lower. These observations appear to relate adenylate cyclase to a number of other anion activated enzymes, some of which have already been studied in pure form by a number of physico-chemical techniques, and which may serve as models for understanding the action of fluoride and other anions on adenylyl cyclase.     

Micromanipulation of phospholipid bilayers by atomic force microscopy

The molecular details of adhesion mechanics in phospholipid bilayers have been studied using atomic force microscopy (AFM). Under tension fused bilayers of dipalmitoylphosphatidylcholine (DPPC) yield to give non-distance dependent and discrete force plateaux of 45.4, 81.6 and 113±3.5 pN. This behaviour may persist over distances as great as 400 nm and suggests the stable formation of a cylindrical tube which bridges the bilayers on the two surfaces. The stability of this connective structure may have implications for the formation of pili and hence for the initial stage of bacterial conjugation. Dimyristoylphosphatidylcholine (DMPC) bilayers also exhibit force plateaux but with a much less pronounced quantization. Bilayers composed of egg PC, sterylamine and cholesterol stressed in a similar way show complex behaviour which can in part be explained using the models demonstrated in the pure lipids.     

Clustering of fatty acids in phospholipid bilayers.

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at pH greater than 11 is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Mitophagy in yeast occurs through a selective mechanism

The regulation of mitochondrial degradation through autophagy is expected to be a tightly controlled process, considering the significant role of this organelle in many processes ranging from energy production to cell death. However, very little is known about the specific nature of the degradation process. We developed a new method to detect mitochondrial autophagy (mitophagy) by fusing the green fluorescent protein at the C terminus of two endogenous mitochondrial proteins and monitored vacuolar release of green fluorescent protein. Using this method, we screened several atg mutants and found that ATG11, a gene that is essential only for selective autophagy, is also essential for mitophagy. In addition, we found that mitophagy is blocked even under severe starvation conditions, if the carbon source makes mitochondria essential for metabolism. These findings suggest that the degradation of mitochondria is a tightly regulated process and that these organelles are largely protected from nonspecific autophagic degradation.