Electron paramagnetic resonance studies on the fluidity and surface dynamics of egg phosphatidylcholine vesicles containing gangliosides.

The influence of different gangliosides (GM1, GD1a, GT1b) on the fluidity and surface dynamics of phosphatidylcholine small unilamellar vesicles was studied by electron paramagnetic resonance. 5- and 16-nitroxystearic acid, sounding respectively the region close to the surface and that close to the hydrophobic core of the vesicle, were employed as spin-label probes. The signals released by 5-nitroxystearic acid showed that the presence of gangliosides reduced the mobility of the hydrocarbon chains around the probe. The effect increased by increasing ganglioside concentration, and diminished from GM1 to GD1a and GT1b. The decrease of membrane fluidity was also monitored by the 16-nitroxystearic acid probe. On addition of Ca2+ the fluidity of ganglioside-containing vesicles (as signalled by the 5-nitroxystearic acid probe) promptly decreased, therefore returning slowly to the original value. It is suggested that gangliosides cause strong side-side head group interactions on the bilayer surface--between ganglioside oligosaccharide chains and between ganglioside and phosphatidylcholine polar portions--which lead the lipid chains to assembly in a more rigid fashion. The influence of Ca2+ is interpreted as due to lateral phase separation in the vesicle membrane. This phenomenon can be related to the formation or stabilization of ganglioside clusters on the vesicle surface.

A new fluorescence method to detect singlet oxygen inside phospholipid model membranes.

A fluorescence method for detecting singlet oxygen (1O2) in model membranes is proposed. 1O2 was generated by hydrogen peroxide/sodium hypochlorite system. 1,3-Diphenylisobenzofuran (DPBF), a specific 1O2 trap, dissolved in organic solvents gives a strong fluorescence spectrum when excited at 410 nm. A similar spectrum, with a maximum at 455 nm, is obtained when DPBF is incorporated in unilamellar dipalmitoylphosphatidylcholine liposomes. The intensity of fluorescence spectrum decreases when DPBF-labeled liposomes are exposed to singlet oxygen. This decrease is sensitive to 1O2 traps and quenchers like tryptophan and sodium azide, to lipid membrane fluidity and to the concentration of sodium hypochlorite and hydrogen peroxide.

Effect of neutral and acidic phospholipids on mitochondrial ATP synthase secondary structure.

The secondary structure of delipidated and egg phosphatidylcholine or asolectin reconstituted mitochondrial ATP synthase complex from beef heart was investigated by Fourier transform infrared spectroscopy. Upon reconstitution, the infrared spectra of ATP synthase revealed an increase in turns and a concomitant decrease in beta-sheet content which occurred to a larger extent in the presence of asolectin rather than in the presence of egg phosphatidylcholine. These data correlate with kinetic data showing a higher ATPase activity of the asolectin reconstituted enzyme protein than the egg phosphatidylcholine reconstituted or delipidated enzyme complexes.

Steady-state fluorescence anisotropy and multifrequency phase fluorometry on oxidized phosphatidylcholine vesicles.

Multilamellar liposomes, from mixtures of unoxidized (control) and singlet oxygen oxidized phosphatidylcholine, were studied by steady-state fluorescence anisotropy and multifrequency phase fluorometry using 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent probe. Lifetime fluorescence decay of the DPH-labeled liposomes was analyzed either by a model of discrete exponential components and a model that assumes a continuous distribution of lifetime values. Increasing the oxidized phosphatidylcholine content in the liposomes, an increase of the membrane interior polarity and a decrease of membrane fluidity occurs which can be related to the hydroperoxide-lipids and double bonds conjugation, respectively.

Effect of the fungicides tributyltin acetate and tributyltin chloride on multilamellar liposomes: fluorescence studies.

The influence of tri-n-butyltin acetate (TBTA) and tri-n-butyltin chloride (TBTC) on the physico-chemical state of charged and neutral phospholipids was investigated using multilamellar liposomes. The thermal dependence of steady state fluorescence polarization of DPH and its charged derivative TMA-DPH was recorded. The two fungicides lowered DPPC phase transition temperature and broadened the temperature range of the transition in different ways. The effects were concentration-dependent. The results show that TBTC interacts more effectively with DPPC model membranes rather than TBTA. Moreover, TBTC broadens and shifts the main phase transition (Tm) more effectively in DPPC rather than in DMPC liposomes. Below Tm, TBTC decreases fluorescence polarization (P) in all phospholipids used. Above Tm P is almost constant in phospholipids with saturated acyl chains, except for DMPG. In fact, an increase of P is detectable in this lipid as in PLs with unsaturated acyl chains. It is suggested that the effects of TBT on liposomal membranes are dependent on the anion moiety and phospholipids characteristics.

Interaction of the herbicide atrazine with model membranes. II: Effect of atrazine on fusion of phospholipid vesicles.

The effect of atrazine on Ca2+ induced fusion of cardiolipin(CL) and phosphatidylserine (PS) vesicles is studied by Tb3+/dipicolinic acid fluorescence and turbidity measurements. The interaction of herbicide with CL and PS membranes is studied by DPH fluorescence polarization. At low concentrations the pesticide partially inhibits fusion, especially in CL vesicles. Higher concentrations of atrazine decrease inhibition of fusion in CL, while fusion is slightly increased in PS. The Ca2(+)-induced increase of turbidity is not affected by atrazine in both PS and CL aggregation experiments. DPH polarization measurements show a perturbation only of the membrane hydrophobic core of PS, in presence of Ca2+. It is hypothesized that this biphasic effect shown by low and high atrazine concentrations on Ca2(+)-induced fusion of vesicles is due to a different localization of the pesticide in the membrane.

Effect of N-acylethanolamines with different acyl-chains on DPPC multilamellar liposomes.

The influence of N-acylethanolamines with different acyl-chains on the physico-chemical state of neutral phospholipids was investigated using dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes. The thermal dependence of steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its charged derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was recorded. The N-acylethanolamines modified the DPPC phase transition temperature and broadened the transition temperature range in different ways depending on the N-acylethanolamines acyl chain characteristics. Our data suggest that the N-acylethanolamine acyl chain length and unsaturation play an important role in the interaction of these compounds with model membranes. The results show that long-chain-N-acylethanolamines interact largely with DPPC model membranes while a similar effect is not observed for the short ones.

Interaction of the herbicide atrazine with model membranes. I: Physico-chemical studies on dipalmitoyl phosphatidylcholine liposomes.

Atrazine (2-chloro-4 ethylamino-6-(isopropylamino)-s-triazine) is one of the most widely used herbicides. Fourier transform infrared spectroscopy, differential scanning calorimetry and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were used to study the interaction of atrazine with dipalmitoyl phosphatidylcholine liposomes used as a model for biological membranes. The results show that atrazine does not perturb the hydrophobic core of the lipid bilayer and suggest that the herbicide localizes near the glycerol backbone of the lipid.

Permeability of oxidized phosphatidylcholine liposomes.

Permeability of liposomes made from mixtures of unoxidized and singlet oxygen oxidized phosphatidylcholine has been related to the degree of lipid oxidation expressed as hydroperoxide moiety content in the lipids. The effect of oxidation on the liposomes permeability has been studied by fluorometry using calcein as a fluorescent probe that undergoes self quenching when highly concentrated inside liposomes. The liposomes containing 73% and 5% of hydroperoxides retain respectively 64.5 and 96.3% of calcein with respect to that retained by the liposomes made from unoxidized phosphatidylcholine. The fluorescence data show a linear relationship between the liposome permeability and the oxidation degree of lipids.

Differential scanning calorimetry characterization of oxidized egg phosphatidylcholine liposomes.

Differential scanning calorimetry has been used to characterize liposomes made from mixtures of unoxidized and singlet oxygen oxidized egg phosphatidylcholine. Cooling scans reveal that trapped water decreases when the oxidized phosphatidylcholine content is increased in the liposomes. Liposomes made from mixtures containing more than 50% by weight of oxidized phosphatidylcholine do not show trapped water.

Effect of some lipophilic substances on fluorescence polarization of perylene in lipid vesicles and mitochondrial membranes.

Short-chain ubiquinones were observed to increase the fluorescence polarization associated with perylene, indicating a decrease in the fluidity of the mitochondrial membrane. The results indicated that the perturbation induced by low homologs results indicated that the perturbation induced by low homologs of ubiquinone on the physical state of membrane lipids is quite different from that of other lipophilic substances which have been considered.

HtrA heat shock protease interacts with phospholipid membranes and undergoes conformational changes.

The HtrA (DegP) protein of Escherichia coli is a heat shock serine protease, essential for cell survival only at temperatures above 42 degrees C. It has been shown by genetic experiments that HtrA is an envelope protease, functioning in the periplasmic space. To clarify the cellular localization of HtrA, E. coli cells were fractionated, and HtrA was not detected by the immunoblotting technique in the periplasm or in the fraction of soluble proteins but was found in the inner membrane. The protein could be partially eluted from the total membrane fraction by a high ionic strength solution, whereas solutions affecting protein conformation released HtrA almost completely. These results, taken together with the evidence showing that HtrA functions in the periplasm, indicate that HtrA is a peripheral membrane protein, localized on the periplasmic side of the inner membrane. As the first step toward solving the problem of HtrA-membrane interactions, the structure of HtrA in the presence of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), or cardiolipin (CL) was analyzed by fluorescence and Fourier-transform infrared spectroscopy. The infrared and fluorescence data indicated an interaction of HtrA with PG and CL but not with PE suspensions. Fluorescence spectroscopy revealed that this interaction was at the level of the polar head group of the phospholipid. In the PG/HtrA system, small changes were observed in the HtrA secondary structure and a remarkable decrease of the thermal stability of the protein, which suggested changes in HtrA tertiary structure. This suggestion was supported by fluorescence data that showed a shift of the fluorescence emission spectrum of HtrA tyrosine residues in the presence of PG and a reduced fluorescence intensity, phenomena not observed in the presence of PE or CL suspensions. Infrared data revealed also that the interaction of HtrA with PG leads to a protection of unfolded protein against aggregation at relatively low temperatures. The conformational changes of HtrA in the presence of PG influenced the proteolytic activity of HtrA by increasing it at the temperatures 37-45 degrees C and inhibiting it at 50-55 degrees C. CL inhibited HtrA activity at all of the temperatures tested.

Physicochemical characterization of plasma membranes from density-separated trout erythrocytes.

Erythrocytes of Salmo irideus trout were separated in the range from 45 to 65% Percoll, yielding three well-separated different fractions. Steady-state fluorescence of probes embedded in erythrocyte membranes and/or in liposomes from extracted lipids was used to characterize their physicochemical properties. Furthermore, the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), embedded in the same liposomes, was measured by a frequency decay fluorometer. DPH decay was analyzed on the assumption of continuous distribution of lifetimes, for evaluating modifications of membrane microheterogeneity. Significant differences were observed in the parameters measured for the three erythrocyte fractions, possibly connected with the specific lipid composition of the samples.

Specific interaction of cytosolic and mitochondrial glyoxalase II with acidic phospholipids in form of liposomes results in the inhibition of the cytosolic enzyme only.

Kinetics of cytosolic recombinant human glyoxalase II and bovine liver mitochondrial glyoxalase II were studied in the presence of liposomes made of different phospholipids (PLs). Neutral PLs such as egg phosphatidylcholine or dipalmitoylphosphatidylcholine did not affect the enzymatic activity of either enzymatic form. Liposomes made of dioleoyl phosphatidic acid or cardiolipin or phosphatidylserine also did not affect the enzymatic activity of mitochondrial glyoxalase II. Conversely, these negatively charged PLs exerted noncompetitive inhibition on cytosolic glyoxalase II only, dioleoyl phosphatidic acid and bovine brain phosphatidylserine exerting the highest and lowest inhibition, respectively. Binding studies, carried out by using a resonant mirror biosensor, revealed that liposomes made of negatively charged PLs interact specifically with both enzymatic forms of glyoxalase II, whereas interactions were not detected with neutral PLs. Once bound on glyoxalase II, negatively charged liposomes could not be removed by 3 M NaCl, suggesting that interactions between glyoxalase II and negatively charged PLs, besides ionic, may be also hydrophobic. These data suggest a possible role of negatively charged phospholipids in the regulation of level of lactoylglutathione in the cell. The data are also discussed in terms of a possible regulation of reduced glutathione supply to mitochondria.