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P F Devaux

Publications and source records attributed to P F Devaux.

At least 91 records · Page 5Linked to original sources
PubMed1984

Asymmetric lipid fluidity in human erythrocyte membrane: new spin-label evidence.

We have synthesized spin-labeled analogues of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine with a short beta chain (C5) bearing a doxyl group at the fourth position. When added to an erythrocyte suspension, the labels immediately incorporate in the membrane. The orientation of the spin-labels was assessed in the bilayer (i) by addition in the medium of a nonpermeant reducer (ascorbate at 5 degrees C) or (ii) by following spontaneous reduction at 37 degrees C due to the endogenous reducing agents present in the cytosol. Both techniques prove that the spin-labels are originally incorporated in the outer leaflet and redistribute differently after incubation. After a 5-h incubation at 5 degrees C, the phosphatidylcholine derivative remained in the outer layer, while the phosphatidylethanolamine and phosphatidylserine derivatives were found principally in the inner leaflet. During the incubation, a small fraction of the spin-labels is hydrolyzed, particularly the phosphatidylserine derivative, presumably by an endogenous phospholipase A2. Because the hydrolyzed spin-labeled fatty acids are rejected in the aqueous phase, the spectra of the intact membrane-bound phospholipids can be obtained by an adequate spectral subtraction. The ESR spectrum corresponding to a probe in the outer leaflet indicates a more restricted motion than that associated with probes in the inner leaflet. Additional experiments have been carried out to prove that the difference in viscosity, which is likely to be due to anisotropic cholesterol distribution, is not attributable to modification of the cell morphology.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Spin Resonance Spectroscopy↗
PubMed1984

Spin-label studies of the oligomeric structure of band 3 protein in erythrocyte membranes and in reconstituted systems.

A spin-labeled fatty acid (16-doxylstearic acid), linked by an ester bond to a maleimide or a nitrene residue, was covalently attached to band 3 of erythrocyte membranes. The electron spin resonance spectrum of the spin-labeled protein was examined at different temperatures in: (a) whole erythrocyte ghosts; (b) ghosts depleted of spectrin and actin; (c) alkaline-treated ghosts; (d) vesicles made with purified band 3 reassociated with dimyristoylphosphatidylcholine. Most spectra are composite with a major component corresponding to a large overall splitting. The determination of the percentage of the immobilized component was carried out by pairwise subtraction. At low temperatures (1-7 degrees C), the highest fraction of immobilized component was found in dimyristoylphosphatidylcholine vesicles (approx. 100%); alkaline-treated membranes had approx. 75% of the immobilized component at the same temperature; whole erythrocyte, spectrin/actin-depleted and spectrin/actin/ankyrin-depleted ghosts gave identical results (approx. 60% of immobilized component). The immobilized fraction decreased in all samples with increasing temperature or addition of a nonsolubilizing concentration of dodecyl octaethylene glycol monoether. In dimyristoylphosphatidylcholine vesicles, however, the modification in the ration of the two components was obtained only above the lipid transition temperature (23 degrees C). The strong immobilization of the spin-labeled lipid chain at all temperatures suggested trapping of the lipid chain between proteins. At low temperature, in dimyristoylphosphatidylcholine vesicles or in alkaline-treated ghosts, lipid-protein segregation is likely to take place. In whole erythrocyte ghosts, on the other hand, the large contribution of the motionally restricted component at physiological temperature indicates the oligomeric nature of band 3. Partial dissociation of the oligomers occurs as the temperature is increased, but the presence or absence of cytoskeletal proteins has no influence on the state of oligomerization of band 3.

Actins↗
PubMed1984

ATP-dependent asymmetric distribution of spin-labeled phospholipids in the erythrocyte membrane: relation to shape changes.

Spin-labeled analogs of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine have been used to study phospholipid transverse diffusion and asymmetry in the human erythrocyte membrane. Ascorbate reduction was used to assess the transbilayer distribution of the labels. All three spin-labeled phospholipids initially incorporated into the outer leaflet of the membrane. On fresh erythrocytes at 5 degrees C, the phosphatidylcholine label remained mainly in the outer leaflet. In contrast, the phosphatidylserine and phosphatidylethanolamine labels underwent rapid transverse diffusion that led to their asymmetric distribution in favor of the inner leaflet. The latter effect was reversibly inhibited after ATP depletion of the erythrocytes and could be reproduced on resealed erythrocyte ghosts only if hydrolyzable Mg-ATP was included in the internal medium. It is suggested that an ATP-driven transport of amino phospholipids toward the inner leaflet could be the major cause of the phospholipid asymmetry in the erythrocyte membrane. It is also proposed that the same mechanism could explain the ATP requirement of the maintenance of the erythrocyte membrane discoid shape.

Adenosine Triphosphate↗
PubMed1983

Non-uniform distribution of phospholipids in (Na+ + K+)-ATPase-rich membranes from Torpedo marmorata electric organ evidenced by spin-spin interactions between spin-labeled phospholipids.

(Na+ + K+)-ATPase membranes from Torpedo marmorata electric organ were labeled with high concentrations of paramagnetic phospholipid analogs (up to 5 mol of spin labels for 100 mol of endogeneous phospholipids). The mobile lipid bilayer component of the complex resonance spectra obtained showed low-field linewidth broadening due to spin-spin interactions; this was used as a measure of probe concentration in this compartment. Our results show that in the lipid shell surrounding the protein, there is a considerable enrichment in negatively charged phospholipids over neutral ones.

Animals↗
PubMed1983

Collisions between nitrogen-14 and nitrogen-15 spin-labels. 1. Lipid-lipid interactions in model membranes.

A new method is described to evaluate collision frequencies between spin-labeled molecules in membranes. This method utilizes a mixture of 14N and 15N nitroxides. The extent of spin-spin interaction between the two different isotopes can be determined by comparing experimental ESR spectra with simulated spectra. The latter are calculated by using modified Bloch equations and by assuming that the spin exchange process is the dominant broadening mechanism. A more approximate analysis is also described; it is based on the evaluation of the line broadening from the measurement of peak heights. Both analyses show that relatively low concentrations of label can be utilized: 1-5% labeled lipids in a membrane give a broadening sufficient to allow quantitation. Collision frequencies equal to or larger than 0.1 MHz can be evaluated by this method. Because of the limited overlap between 15N and 14N spectra, this method allows the investigation of the collisions between unlike molecules of very different concentration. From the collision frequencies, the topological domain explored by a probe can be inferred as well as its lateral diffusion constant. In this paper, it is shown that in spite of the low order of the chain residues near their methyl terminal, a probe linked at the omega - 2 position of an ionized stearic acid does not interact with a probe positioned near the polar region. Nevertheless the former probe, deeply embedded in the membrane, explores a larger volume. It is also shown that if the nitroxide is attached to a fatty ester, the probe "sees" evenly the whole hydrophobic region of the membrane.

Chemical Phenomena↗
PubMed1983

Collisions between nitrogen-14 and nitrogen-15 spin-labels. 2. Investigations on the specificity of the lipid environment of rhodopsin.

The method of spin-spin interactions between 15N and 14N spin-labels was used to investigate lipid-protein collision rates in reconstituted vesicles containing rhodopsin from bovine disk membranes and an equimolar mixture of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In each sample, a fraction of one of the three phospholipids was labeled with 14N spin-label while a 15N spin-labeled fatty acid was covalently linked to rhodopsin. The extent of spin-spin interaction between 15N and 14N labels was either calculated by complete spectral simulation or evaluated from the line broadening as deducted from the intensity decrease of the low-field 15N line. It was found that all three spin-labeled phospholipids utilized for these experiments can interact magnetically with the spin-labeled rhodopsin. Above 35 degrees C little difference between the three species can be detected. Calculation of the diffusion constant of the phospholipids at the boundary of rhodopsin proves that the lifetime of the phospholipids at the protein boundary is short and that no long-lived annular lipids are segregated. At temperatures below approximately 30 degrees C the spectra of the samples containing spin-labeled phosphatidylserine depend upon the presence or absence of calcium. The extent of 15N line broadening was found weaker in the presence of Ca2+ than in the presence of ethylenediaminetetraacetate. Thus Ca2+ tends to exclude phosphatidylserine from the lipid environment of rhodopsin. This observation can be attributed to the formation of specific lipid domains within the membrane, induced by Ca2+.

Animals↗
PubMed1982

Evidence for protein-associated lipids from deuterium nuclear magnetic resonance studies of rhodopsin-dimyristoylphosphatidylcholine recombinants.

The technique of deuterium magnetic resonance was used to study the orientational order of the perdeuterated acyl chains of dimyristoylphosphatidylcholine (DMPC-d54) reconstituted with rhodopsin between 0 and 23 degrees C. This range includes the gel to liquid crystalline phase transition of DMPC-d54 at 20 degrees C. Molar lipid/protein (L/P) ratios of L/P = infinity, 150, 50, 30, and 12 were investigated. Measurements of orientational order parameters showed that the addition of rhodopsin broadened the range of the gel to liquid crystalline transition for L/P = 150 and 50. No transition was observed for L/P = 30 and 12. Moment analysis and spectral subtraction both showed that the low temperature spectra for L/P greater than 30 had two components. One was a pure phospholipid gel phase spectrum and the other a spectrum attributed to lipids in protein aggregates. The intensity of the second component corresponded to 30 lipids/protein and its shape was the same as the temperature-independent shape observed for L/P = 30 and 12. No such decomposition into two components was possible in the liquid crystalline phase for L/P greater than 30. Extraction of the oriented 2H NMR spectrum from its powder spectrum showed that the presence of proteins does not modify the distribution of quadrupole splittings but does produce both homogeneous and inhomogeneous broadening. The latter may be related to the heterogeneity seen in the spectra of electron paramagnetic resonance spin labels above the gel to liquid crystalline transition.

Dimyristoylphosphatidylcholine↗
PubMed1982

Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules.

The conventional method of studying mass transport in membranes by spot photobleaching and then following the recovery of fluorescence has disadvantages. Among them, the need for a high density of fluorescent molecules, the measurement of the beam profile, and a knowledge of the photobleaching processes are of a crucial importance. The application of a planar fringe pattern of light both for the bleaching and the monitoring of the fluorescent molecules solves these three major difficulties. Brownian diffusion coefficients and flow velocities can be measured independently and are averaged over the whole fringe pattern volume. These transport coefficients are explored over the wide range of experimentally accessible distances (from an interfringe spacing 0.5-50 micron). The quantification of the mobile and immobile components is further simplified by scanning the fringe pattern and detecting only a modulated fluorescence recovery signal. The fringe pattern photobleaching method is particularly adapted to the measurements of diffusion coefficients and flow velocity of membrane components, as well as of cytoplasmic proteins. The theoretical results and the test experiments with fluorescent bovine serum albumin are described.

Animals↗
PubMed1982

The rotational diffusion of the acetylcholine receptor in Torpeda marmorata membrane fragments studied with a spin-labelled alpha-toxin: importance of the 43 000 protein(s).

The rotational diffusion of the acetylcholine (ACh) receptor in subsynaptic membrane fragments from Torpedo marmorata electric organ was investigated with a spin-labelled alpha-bungarotoxin. A toxin with two spin labels was first synthesized; the conventional electron spin resonance spectrum (e.s.r.) of this toxin bound to the receptor indicated: (1) a complete immobilization of the probes; and (2) a strong spin-spin interaction that was not, or barely, seen in solution. The modification of the degree of spin-spin interaction is taken as an indication of a toxin conformational change accompanying its binding to the ACh-receptor. To avoid spin-spin interaction a single-labelled toxin was made and used to follow the rotational diffusion of the receptor by saturation transfer e.s.r. (ST-e.s.r.). With native membranes a high immobilization of the ACh-receptor was noticed. Reduction of the membranes by dithiothreitol had little effect on this motion. Only extraction of the 43 000 protein(s) by pH 11 treatment was able to enhance the rotational diffusion of the ACh-receptor protein (rotational correlation time by ST-e.s.r. in the 0.5 - 1 X 10(-4) s range) and to allow its lateral diffusion in the plane of the membrane fragments (observed by electron microscopy after freeze-etching or negative staining).

Animals↗
PubMed1981

Effects of temperature, lipid modification and pH on the mobility of the major proteins of the receptor-rich membranes from Torpedo marmarata.

The factors influencing the overall mobility of the major proteins of the acetylcholine receptor-rich membranes from Torpedo marmorata have been investigated by saturation transfer ESR spectroscopy and the lateral distribution of these proteins has been studied by electron microscopy. A spin-labelled derivative of maleimide, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MSL), was used under various conditions of incubation, enabling us to attach it mainly to either an extrinsic protein of 43 kdaltons, or an intrinsic protein (40 kdaltons) bearing the alpha-toxin-binding site. (1) The direct reaction of MSL with the membrane fragments resulted in almost exclusive labelling of the 43 kdalton protein, an extrinsic protein located on the inner face of the receptor-rich membranes. (2) After the free SH groups were blocked with N-ethylmaleimide and the disulfide bridges opened with the reducing agent dithiothreitol, MSL reacted with both the 40 and 43 kdalton proteins (6.0 +/- 0.6 MSL molecules per alpha-toxin-binding site). (3) After the latter labelling procedure membranes were exposed to pH 11, resulting in extraction of the 43 kdalton protein and leaving 2.2 +/- 0.4 MSL molecules per alpha-toxin-binding site; sodium dodecyl sulfate polyacrylamide gel electrophoresis performed with N-[14C] ethylmaleimide suggested that MSL was bound mainly to the 40 kdalton polypeptide chain of the acetylcholine receptor. The following conclusions were made with the native and alkaline-treated membranes: In the native membranes, saturation transfer ESR does not reveal any significant protein rotational diffusion (rotational correlation time tau C greater than 1 ms). Temperature variations and/or lipid modifications obtained by fusion of exogenous lipids and/or cholesterol exchange have little influence on the saturation transfer ESR spectra. Electron microscopy reveals that upon lipid addition, proteins remain in the form of clusters while areas depleted of proteins appear. On the other hand, alkaline treatment strikingly enhances the motion of the MSL-labelled proteins in the membrane (100 less than or equal to tau c less than or equal to 120 microseconds). Furthermore, the rotational diffusion of the MSL-labelled proteins (mainly the 40 kdalton protein) becomes sensitive to temperature, lipid composition and the lipid-to-proteins ratio. Electron microscopy shows that alkaline extraction does not cause large reorganization of the acetylcholine receptor in the plane of the membrane. However, when phospholipids are added to pH 11 treated membranes, a dispersion of the receptor and rosettes is observed. In contrast, cholesterol enrichment of the latter membranes induces clustering of the receptor immobilization as judged by saturation transfer ESR. Upon reassociation of the pH 11 soluble proteins with the alkaline-treated membranes, the restriction of the acetylcholine receptor rotational mobility is also restored (tau c greater than or equal to 1 ms).

Animals↗
PubMed1981

Distinct states of lipid mobility in bovine rod outer segment membranes. Resolution of spin label results.

Freely diffusable lipid spin labels in bovine rod outer segment disc membranes display an apparent two-component ESR spectrum. One component is markedly more immobilized than that found in fluid lipid bilayers, and is attributed to lipid interacting directly with rhodopsin. For the 14-doxyl stearic acid spin label this more immobilized component has an outer splitting of 59 G at 0 degrees C, with a considerable temperature dependence, the effective outer splitting decreasing to 54 G at 24 degrees C. Spin label lipid chains covalently attached to rhodopsin can also display a two-component spectrum in rod outer segment membranes. In unbleached, non-delipidated membranes the 16-doxyl stearoyl maleimide label shows an immobilized component which has an outer splitting of 59 G at 0 degrees C and a considerable temperature dependence. This component which is not resolved at high temperatures (24--35 degrees C), is attributed to the lipid chains interacting directly with the monomeric protein, as with the diffusable labels. In contrast, in rod outer segment membranes which have been either delipidated or extensively bleached, a strongly immobilized component is observed with the 16-doxyl maleimide label at all temperatures. This immobilized component has an outer splitting of 62--64 G at 0 degrees C, with very little temperature dependence (61--62 G at 35 degrees C), and is attributed to protein aggregation.

Animals↗
PubMed1981

Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ.

After fixation with glutaraldehyde and impregnation with tannic acid, the membrane that underlies the nerve terminals in Torpedo marmorata electroplaque presents a typical asymmetric triple-layered structure with an unusual thickness; in addition, it is coated with electron-dense material on its inner, cytoplasmic face. Filamentous structures are frequently found attached to these "subsynaptic densities." The organization of the subsynaptic membrane is partly preserved after homogenization of the electric organ and purification of acetylcholine-receptor (AchR)-rich membrane fragments. In vitro treatment at pH 11 and 4 degrees C of these AchR-rich membranes releases an extrinsic protein of 43,000 mol wt and at the same time causes the complete disappearance of the cytoplasmic condensations. Freeze-etching of native membrane fragments discloses remnants of the ribbonlike organization of the AchR rosettes. This organization disappears ater alkaline treatment and is replaced by a network which is not observed after rapid freezing and, therefore, most likely results from the lateral redistribution of the AchR rosettes during condition of slow freezing. A dispersion of the AchR rosettes in the plane of the membrane also occurs after fusion of the pH 11-treated fragments with phospholipid vesicles. These results are interpreted in terms of a structural stabilization and immobilization of the AchR by the 43,000-Mr protein binding to the inner face of the subsynaptic membrane.

Animals↗