The work reported herein deals with the evaluation of the antioxidant properties of bitailed amphiphilic α-phenyl-N-tert-butylnitrone derivatives (BPBNs) towards oxidation of an unsaturated lipid, the 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC). Oxidation was induced either by UV light irradiation or radical initiators, i.e. the water soluble AAPH and the Fenton reaction, and the antioxidant evaluation was carried out using two biomimetic systems, namely Langmuir monolayers and large unilamellar vesicles. Measurement of the molecular area and the membrane fluidity of pure nitrone monolayers before and after UV-irradiation demonstrated the better stability and antioxidant properties of B17PBN, the derivative with two C17H35 alkyl chains, compared to its analogue B11PBN with two C11H23 alkyl chains. At only 5% molar ratio of nitrone in mixed DLoPC/nitrone monolayers, a complete inhibition of the molecular area decrease was observed for B17PBN whereas B11PBN showed lower protection. The oxidation of mixed DLoPC/nitrones large unilamellar vesicles in the presence of free radicals arising from AAPH decomposition or Fenton reaction was assessed by measuring lipid conjugated dienes and thiobarbituric acid reactive substances on the whole series of nitrone, i.e. C11-, C13-, C15- and C17-based compounds. Compared to the saturated 1,2-dimyristoyl-sn-glycero-3-phosphocholine, all bitailed amphiphilic nitrones were able to decrease conjugated dienes and TBARS in both oxidative paradigms, demonstrating therefore antioxidant property. The inhibition of phospholipids oxidation was increased when increasing the concentration of nitrone with the two B11PBN and B13PBN derivatives exhibiting higher potency. This study underlines the importance in the choice of a model membrane system when evaluating the potency of antioxidants against lipid oxidation.

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential. On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments. When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone H1 and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.

Glycosylphosphatidyl inositol (GPI) anchored proteins are localized and clustered on the outer layer of the plasma membranes forming microdomains. Among them, mammalian alkaline phosphatases (AP-GPI) are widely distributed enzymes. They can also exist as soluble proteins without anchor (APs). Using the Langmuir film technique, we study the thermodynamic properties of monolayers for both protein forms at the air-buffer interface. The enzymatic activity is maintained at the interface but the adsorption of the two forms of AP is very different. AP-GPI presents a higher surface activity and a larger molecular area than the soluble form. The molecular area deduced for high surface pressures suggests a different organization of the monolayers for these two forms. APs molecules seem to adsorb as a multilayer at the interface while AP-GPI appear to be orientated with the major axis parallel to the interface. This orientation allows the accessibility of AP-GPI enzymatic sites that are turned in direction of the subphase as in vivo where the active sites must be turned outside of the membrane.

Mammalian alkaline phosphatases (AP) are glycosylphosphatidylinositol (GPI) anchored proteins that are localized on the outer layer of the plasma membrane. The GPI anchors are covalently attached to the C-termini of proteins and consist of a glycan chain bonded to phosphatidylinositol with two acyl chains anchored into the membrane bilayer. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the adhesion of alkaline phosphatase in the absence and presence of anchors. The GPI anchors increase markedly the adhesion frequency (i.e., the protein affinity for the membrane). An adhesion force of 350 +/- 200 pN is measured between GPI-anchored AP (AP(GPI)) and supported phospholipid bilayers of dipalmitoylphosphatidylcholine (DPPC) presenting structural defects (holes). In the absence of defects, the adhesion force (103 +/- 17 pN) and the adhesion frequency are reduced. These results indicate that AP(GPI) poorly spontaneously insert into membranes in vivo and open new perspectives for the characterization of the interactions between GPI proteins and membranes.

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of (3)H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance-about 2.5 nm-between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein.

The interfacial behavior differences of two glutathione peroxidase isoforms have been investigated. The first isoform is the phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12) (GPx-4) isolated from rat testes and the second one is the cytosolic glutathione peroxidase (EC 1.11.1.9) (GPx-1) from bovine erythrocytes. Injected in the subphase buffer of a Langmuir trough, GPx-4 was able to adsorb quickly at the air-water interface whereas the GPx-1 was not. Then, the protein interaction with phospholipid monolayers was explored. Indeed, a monolayer of phospholipids containing a different number of polyunsaturated fatty acyl chains was prepared at the air-water interface. Under each kind of monolayer, the protein solution was injected and its adsorption was visualized by the measurement of successive pressure-area isotherms. We have, then, determined the molecular area increase due to the protein adsorption. It was found that the GPx-4 is adsorbed in each kind of monolayer tested whereas no molecular area increase was detected with the GPx-1. This indicates that the GPx-4 has a higher affinity for the interface, recovered or not by lipids, than the GPx-1. Moreover, the GPx-4 presents a different affinity for the phospholipid monolayers depending on the number of polyunsaturated fatty acyl chains.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed, to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into Supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.

decapeptides with a lactone linkage between the carboxyl group of the C-terminal residue and the phenyl hydroxyl of Tyr3. PL were isolated from Bacillus cereus as an inhibitor of phospholipase A2, but they also inhibit phospholipase D from cabbage2). The effects of SF or BL on phospholipases D (PLD) have not been yet studied. It was only shown that SF inhibits cytosolic phospholipases A23). In this study, on the contrary of the previous studies on phospholipase inhibitors2,3), we used a soluble substrate of phospholipase D from S. chromofuscus (PLDsc). In these conditions, only the PLDsc inhibition by the lipopeptides would be studied and possible interactions between the lipopeptides and the phospholipidic substrate (i.e. liposomes) would be excluded. More precisely, we investigated the effects of PL, BL and SF on PLDsc by measuring its activity by using a rapid method involving bis(para-nitrophenyl)phosphate [bis(pNP)P]4). PLDsc was purchased from Sigma Chemical Co. (St. Louis, MO) and lipopeptides were prepared from B. subtilis cultures as described in References1,5,6). The influence of SF, BL and PL on PLDsc activity was

The phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme that interacts with membranes to catalyse phosphatidylcholine (PC) transformation. In this work, we focused on the interaction between PLDSc and two lipid activators: a neutral lipid, diacylglycerol (DAG), and an anionic one, phosphatidic acid (PA). DAG is a naturally occurring alcohol, so it is a potent nucleophile for the transphosphatidylation reaction catalysed by PLD. Concerning PA, it is a widely described activator of PLDSc-catalysed hydrolysis of PC. The monolayer technique allowed us to define PLDSc interaction with DAG and PA. In the case of DAG, the results suggest an insertion of PLDSc within the acyl chains of the lipid with an exclusion pressure of approximately 45 mN/m. PLDSc-DAG interaction seemed to occur preferentially with the lipid in the liquid-expanded (LE) phase. PLDSc interaction with PA was found to be more effective at high surface pressures. The overall results obtained with PA show a preferential interaction of the protein with condensed PA domains. No exclusion pressure could be found for PLDSc-PA interaction indicating only superficial interaction with the polar head of this lipid. Brewster angle microscopy (BAM) images were acquired in order to confirm these results and to visualise the patterns induced by PLDSc adsorption.

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45°C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.

Detergent-resistant membranes (DRM) were prepared from bovine kidney cortex. The criterion used to test their purification was the increase in the activity of a GPI membrane-anchored protein, the alkaline phosphatase. Its association with specific proteins and lipids was tested. Two successive Triton X-100 treatments followed by purification on sucrose gradient at 4 degrees C were necessary to obtain DRM with a maximum of alkaline phosphatase activity and a typical protein pattern. A third Triton treatment did not alter this DRM composition. Among the enriched protein, we identified, by mass spectrometry, a microsomal dipeptidase, which was GPI membrane-anchored. Protein-kinase activities, mainly serine-kinase, were enriched during the DRM purification. Using the typical FTIR olefinic =C-H bands of the acyl chains, a global decrease in the unsaturation level of DRM lipids was observed as compared with total membranes. Three main phospholipids were identified in DRM. Their fatty acid compositions were determined by gas chromatography and compared with those of total membranes. The most enriched saturated fatty acid was palmitic acid (+44% for phosphatidylethanolamine, +52% for phosphatidylcholine and +49% for sphingomyelin), agreeing with a selection of specific phospholipids among the saturated ones during the DRM purification.

Oxidative stress results from the attack by free radicals of several cellular targets (proteins, DNA and lipids). The cell equilibrium is a direct consequence of the pro-/antioxidant balance. In order to understand the physiological processes involved in oxidative stress, we followed oxidation of unsaturated lipids using a biomimetic system: Langmuir monolayers. The oxidation mode chosen was UV-irradiation and the lipid model was a polyunsaturated phospholipid: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC). The monomolecular film technique was used to measure membrane rheology before and after UV-irradiation. We showed that the UV-irradiation of a DLPC monomolecular film led to a molecular area and surface elasticity modulus decrease that attests to the apparition of new molecular species at the air-water interface. The antioxidant effect of a synthetic plasmalogen (1-O-(1'-(Z)-hexadecenyl)-2-O-oleoyl-sn-glycero-3-phosphocholine or P(PLM)OPE) was tested on the oxidation of DLPC. Indeed, for about 25% mol P(PLM)OPE in mixed DLPC/P(PLM)OPE monolayers, a complete inhibition of the molecular area and the surface elasticity modulus decreases was observed in our experimental conditions. Lower P(PLM)OPE quantities delayed but did not prevent the DLPC oxidation in mixed monolayers.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/P-i ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of beta-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.

The phospholipase D (PLD) from Streptomyces chromofuscus belongs to the superfamily of PLDs. All the enzymes included in this superfamily are able to catalyze both hydrolysis and transphosphatidylation activities. However, S. chromofuscus PLD is calcium dependent and is often described as an enzyme with weak transphosphatidylation activity. S. chromofuscus PLD-catalyzed hydrolysis of phospholipids in aqueous medium leads to the formation of phosphatidic acid. Previous studies have shown that phosphatidic acid-calcium complexes are activators for the hydrolysis activity of this bacterial PLD. In this work, we investigated the influence of diacylglycerols (naturally occurring alcohols) as candidates for the transphosphatidylation reaction. Our results indicate that the transphosphatidylation reaction may occur using diacylglycerols as a substrate and that the phosphatidylalcohol produced can be directly hydrolyzed by PLD. We also focused on the surface pressure dependency of PLD-catalyzed hydrolysis of phospholipids. These experiments provided new information about PLD activity at a water-lipid interface. Our findings showed that classical phospholipid hydrolysis is influenced by surface pressure. In contrast, phosphatidylalcohol hydrolysis was found to be independent of surface pressure. This latter result was thought to be related to headgroup hydrophobicity. This work also highlights the physiological significance of phosphatidylalcohol production for bacterial infection of eukaryotic cells.

The synthesis of new amphiphilic alpha-phenyl-N-tert-butylnitrone (PBN) derivatives bearing two hydrocarbon tails with 17 carbon atoms (compound A(17)) or 11 carbon atoms (compound B-11) is reported. The amphiphilic and morphological properties of these potential antioxidant compounds were investigated at the air-water interface. The analyses were conducted using Langmuir film balance and Brewster angle microscopy. As it is possible to obtain pressure-area (pi-A) isotherms from pure A(17) and B-11, this means that they can form stable monomolecular films at the air-water interface. Monolayers of pure PBN derivatives exhibited different interface behavior. Indeed, along with compression, the B-11 monolayer showed only a liquid-expanded phase and the A(17) monolayer showed a liquid-condensed phase. Brewster angle microscopy showed the presence of condensed domains within the A(17) monolayer. The lateral interactions of these compounds with the polyunsaturated phospholipids, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC) and 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), were evaluated by analysis of 7 A isotherms and excess free energy of mixing. Each PBN derivative was miscible with DLoPC or DLoPE and formed nonideal mixed monolayers at the air-water interface. Only the A(17) molecules exhibited favorable interactions at high surface pressures (over 20 mN/m) with both DLoPC and DLoPE, however. Under these conditions, A(17) should be a better antioxidant than B-11.

Bone alkaline phosphatase with glycolipid anchor (GPI-bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI-bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI-bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X-ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1-phosphate, glucose 6-phosphate, glucose 1,6-bisphosphate by GPI-bALP produced hydroxyapatite in a manner similar to that of beta-glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6-12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI-bALP in calcium-containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI-bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI-bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.

Publisher Summary This chapter presents the anchoring of glycosylphosphatidylinositol (GPI) proteins to liposomes. In vivo , GPI-anchored proteins concentrate exclusively into the exoleaflet of eukaryotic cell plasma membranes, together with cholesterol and most of the glycolipids. Additionally, surface antigens, receptors, and transporters also adopt an orientation relevant to their involvement in communication/exchange functions with the external milieu. As a consequence, the physiological membrane environment of GPI proteins is highly oriented, a characteristic that should be taken into account for reconstitution of biomimetic membranes. The procedure described in the chapter for the controlled ectoplasmic anchoring of a GPI protein on a liposome necessitates the appropriate preparation of biological samples. In this method, the liposomes are not considered a bulk phospholipidic support for the protein but rather a starting material as homogeneous and well defined as possible. Moreover, the purified GPI protein must contain only minute amounts of contaminating lipidic compounds if clean and reproducible results are to be obtained. The procedure of reconstitution consists of destabilization of liposomes by the appropriate use of detergent, incubation with the GPI protein, and elimination of excess detergent by dialysis. These steps are described together with additional comments concerning assessment of correct reconstitution.

Mammalian alkaline phosphatases (AP) belong to glycosylphosphatidyl inositol (GPI) anchored proteins family, which are localised and clustered on the outer layer of the plasma membranes forming microdomains. Using Langmuir film and polarisation modulation infrared reflection absorption spectroscopy (PMIRRAS) techniques, the penetration process of the protein into a phospholipid monolayer have been studied at the air–buffer interface. The penetration of AP-GPI in distearoylphosphatidylcholine monolayers (DSPC) induces a more important surface pressure increase than in dioleoylphosphatidylcholine (DOPC) monolayer. However, the exclusion surface pressure rather similar for both lipids, 20.5 and 22 mN m−1 for, respectively, DSPC and DOPC, indicates that the AP-GPI cannot, in similar conditions, insert by itself into bilayer membranes of either biological or mimetic origin. PMIRRAS suggests that the pure acyl chains perdeuterated DSPC (d70-DSPC) interact with Mg2+ present into the buffer. AP-GPI inserts progressively into the d70-DSPC monolayer changing the environment of phospholipid molecules. Amide I band exhibits α helix and β-sheets components with a predominance of the α helix. The shapes, intensities and positions of the amide I and II bands suggest for the α helix an orientation perpendicular to the interface after a period of molecular reorganisation.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C‐terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI‐anchored alkaline phosphatase (AP) was studied in real‐time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelin–dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl−) were used to mimic the fluid‐ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl− lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.

The adsorption of alkaline phosphatase (AP) from buffer solutions into hydrophilic and hydrophobic methylated glass was studied qualitatively by Tapping Mode Atomic Force Microscopy (TMAFM). Three protein samples were used: soluble AP lacking GPI-anchors (APS); purified AP dimer with two GPI anchors (APGPI); and the same purified protein treated with acetone in order to remove all residual lipids (APGPI-AT). All the preparations have a better affinity for hydrophobic surfaces and only partially maintain their enzymatic activity. The adsorption is larger for APGPI-AT. Adsorption and aggregation of GPI-anchored proteins on hydrophobic surfaces occurs in at least two different phases: large protein clusters adsorb rapidly on the surface (in approx. 5–10 min); and then they slowly rearrange in monolayers. The large cluster adsorption at short times suggests that the anchors favour protein aggregation in solution. APS adsorb as individuals, which aggregate and form two-dimensional dendrites and branched structures after drying. Surfaces with GPI-anchored protein occasionally present dendrites after drying, but never surfaces imaged in solution. It indicates that these structures are formed during drying as a result of a diffusion-limited aggregation process.