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P F Devaux

Publications and source records attributed to P F Devaux.

At least 73 records · Page 4Linked to original sources
PubMed1988

Asymmetric distribution of phospholipids in spectrin-poor erythrocyte vesicles.

We have investigated by electron spin resonance, at 37 degrees C, the outside-inside passage and the equilibrium distribution of spin-labeled phospholipids, respectively, in ATP-containing ghosts, in heat-treated erythrocytes, and in heat-induced vesicles. The heat-treated vesicles were spectrin depleted to approximately 25% of the original content and had lost almost 100% of the other cytoskeletal proteins. Yet the vesicles, as long as they contained ATP, were capable of translocating the aminophospholipids with the same efficiency as the heat-treated erythrocytes, and almost with the same efficiency as ATP-containing ghosts. In the vesicles, sphingomyelin and phosphatidylcholine analogues underwent a very slow transverse diffusion as in native cells. We conclude that spectrin and other cytoskeleton proteins are not major factors for the establishment and maintenance of phospholipid asymmetry in human erythrocytes, which may be chiefly due to the aminophospholipid translocase activity.

Adenosine Triphosphate↗
PubMed1988

Influence of chlorpromazine on the transverse mobility of phospholipids in the human erythrocyte membrane: relation to shape changes.

The influence of chlorpromazine (CPZ) on the transverse mobility of spin-labeled phospholipids incorporated into human erythrocytes was investigated by electron spin resonance. The very slow transverse diffusion of phosphatidylcholine, as well as the absence of transverse mobility of sphingomyelin were not modified even by sublytic concentrations (approximately equal to 1 mM) of CPZ. On the other hand, the rapid outside-inside translocation of the aminophospholipids (Seigneuret and Devaux (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), was slightly hindered in CPZ containing membranes. If the spin-labeled aminolipids were incorporated in erythrocytes and allowed to flip to the inner monolayer before CPZ addition, a fraction of the spin labels (10-15%) flipped back instantaneously from the inner to the outer leaflet, upon incubation with CPZ. Similar experiments carried out with spin-labeled phosphatidylcholine and spin-labeled sphingomyelin showed that a fraction of the spin-labeled choline derivatives flip instantaneously to the inner leaflet if CPZ was added after the spin labels. Addition of lysophosphatidylcholine had no effect on the spin-labeled phospholipid redistribution nor on their transmembrane mobility. We interpret the immediate effect of CPZ addition as being due to a reorganization of the bilayer accompanying the rapid CPZ membrane penetration, phenomenon which is independent of the CPZ effect on the steady-state activity of the 'aminophospholipid translocase', the latter effect being probably a direct CPZ-protein interaction. By comparison of the time course of phosphatidylserine transverse diffusion in control discocyte cells and in CPZ-induced stomatocytes, we infer that the difference in cell shape is not a major factor in the regulation of the active inward transport of aminophospholipids in human erythrocytes.

Chlorpromazine↗
PubMed1988

Phospholipid flippases.

Protein mediated phospholipid translocation through membranes has been observed in rat liver endoplasmic reticulum and in the plasma membrane of erythrocytes as well as in a few other cell membranes. Lipid translocation in plasma membranes is ATP dependent and selectively accumulates aminophospholipids on the inner monolayers.

Adenosine Triphosphate↗
PubMed1988

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine.

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous 2H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.

Deuterium↗
PubMed1988

Measurement of outward translocation of phospholipids across human erythrocyte membrane.

Spin-labeled phospholipids have been used to study the outside----inside and inside----outside transport of phospholipids across the human erythrocyte membrane at 37 degrees C. As already shown, inward transport is much faster for aminophospholipids than for phosphatidylcholine. In addition, we show here that outward transport of the phosphatidylserine and phosphatidylethanolamine analogues is three to four times faster than that of phosphatidylcholine. Magnesium depletion of the erythrocytes considerably decreases the outward rate of both aminophospholipids to values close to that of phosphatidylcholine. These results suggest that the outward aminophospholipid translocation is, at least partly, protein mediated. The protein involved could be identical to the inward Mg-ATP-dependent aminophospholipid carrier.

Algorithms↗
PubMed1987

Ion regulation of phosphatidylserine and phosphatidylethanolamine outside-inside translocation in human erythrocytes.

In previous publications, we have shown, by using spin-labeled derivatives, that the translocation of phosphatidylserine and phosphatidylethanolamine from the outer to the inner monolayer of human erythrocyte membrane is a protein-mediated phenomenon, which requires hydrolisable Mg2+-ATP. The inhibition by intracellular Ca2+ (0.2 microM) or by extracellularly added vanadate (50 microM) was reported (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755; Zachowski, A., Favre, E., Cribier, S., Hervé, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). The present article gives further insight into the effects of intracellular and extracellular ions on the aminophospholipid translocation in human erythrocytes. By measuring the cell ATP concentration, we now show that the inhibitory effect of intracellular calcium on spin-labeled aminophospholipid translocation is partly due to the ATP depletion, which follows the increased consumption by the calcium pump. However, a direct inhibitory effect of cytosolic Ca2+ on the aminophospholipid translocase can be demonstrated by measuring the initial rate of aminophospholipid translocation in the presence of variable amounts of intracellular calcium, at fixed ATP concentrations. Moreover, the transmembrane equilibrium distribution of phosphatidylserine and phosphatidylethanolamine are affected differently by Ca2+: when cytosolic Ca2+ concentration is increased, alteration of phosphatidylethanolamine distribution begins as soon as the inward translocation is affected by Ca2+ (approx. 50 nM), whereas phosphatidylserine distribution remains unchanged within a large inhibitory range of cytosolic Ca2+ concentrations and decreases above 0.2 microM of free Ca2+ within the cytosol. Decrease of the intracellular Mg2+ concentration below its physiological value (approx. 2 mM) results in the inhibition of aminophospholipid inward transport, whereas increase of Mg2+ concentration does not modify this transport. If Mn2+ is substituted for Mg2+, part of the aminophospholipid translocation is maintained, whereas if Co2+ is substituted for Mg2+, the rapid translocation is completely abolished. Concentrations as high as a millimolar of extracellular Ca2+, Mg2+ or Mn2+ have no effect on the aminophospholipid translocation. The less usual cations Cr3+, Fe2+, Cu2+, Sn2+ and Eu3+ are also uneffective. With extracellular Ni2+ or Co2+, some inhibition can be observed, half inhibition by Ni2+ corresponding to 500 microM. Vanadyl (VO2+), on the other hand, is a potent inhibitor of the aminophospholipid translocation when applied on the extracellular surface, half-inhibition being reached around 30 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

Labeling of human erythrocyte membrane proteins by photoactivatable radioiodinated phosphatidylcholine and phosphatidylserine. A search for the aminophospholipid translocase.

We have synthesized radioiodinated photoactivatable phosphatidylcholine (125I-N3-PC) and phosphatidylserine (125I-N3-PS). After incubation with red blood cells in the dark, the labeled PC could be extracted but not the corresponding PS molecule, indicating that the latter was transported by the aminophospholipid translocase, but not the former. When irradiated immediately after incorporation, N3-PS, but not N3-PC, partially blocked subsequent translocation of spin-labeled aminophospholipids. Analysis of probe distribution by SDS-polyacrylamide gel electrophoresis revealed that 125I-N3-PS labeled seven membrane bound components with molecular masses between 140 and 27 kDa: one (or several) of these components should correspond to the aminophospholipid translocase.

Carrier Proteins↗
PubMed1987

Selective outside-inside translocation of aminophospholipids in human platelets.

Spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were added to human platelet suspensions. Due to the partial water solubility of these spin-labeled lipids which possess a relatively short beta-chain (C5), they incorporate rapidly in membranes. The orientation of the spin-labels within the platelet plasma membrane was assessed by following the spontaneous reduction at 37 and 4 degrees C due to endogenous reducing agents present in the cytosol. The rate of spontaneous reduction showed unambiguously that the labels incorporated initially in the outer leaflet of the plasma membrane and that the rate of outside-inside translocation of the aminophospholipids was faster than that of the choline derivatives. For example, at 37 degrees C, the half-time for the transverse diffusion of a phosphatidylcholine analogue was found to be of the order of 40 min, while it was less than 7 min for the phosphatidylserine analogue. At low temperatures, a fraction of the labels gave rise to a strongly immobilized ESR component. This fraction, which corresponded to 20-30% of the initial spin-label concentration, was found resistant to chemical reduction from the inner side of the membrane and also to externally added reducing agents such as ascorbate. Presumably these immobilized lipids are trapped in a gel phase formed in the outer leaflet at 4 degrees C. Cell aging, which depletes the cells of ATP, resulted in the progressive inhibition of the fast transport of the aminophospholipids from the outer to inner leaflet. Treatment of the cells with iodoacetamide completely blocked the transverse diffusion of the spin-labels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1987

Orientation and vertical fluctuations of spin-labeled analogues of cholesterol and androstanol in phospholipid bilayers.

We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol analogue appeared, at least in sonicated vesicles, to experience two opposite orientations in the same monolayer, very likely with a rapid reorientation. The possibility of rapid vertical fluctuations of the sterol molecules within the phospholipid bilayer is also discussed.

Androstanols↗
PubMed1987

Phospholipid outside-inside translocation in lymphocyte plasma membranes is a protein-mediated phenomenon.

We have measured the transbilayer diffusion of spin-labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in pig lymphocyte plasma membrane. At 4 degrees C and 37 degrees C the aminophospholipids are rapidly transported from the outer to the inner leaflet of the membrane, whereas the choline-containing phospholipids experience a slower diffusion. This selectivity is abolished after cell treatment by SH-group reagents indicating that the aminophospholipid translocation is protein-dependent and must be driven by a system analogous to the one existing in the human red cell membrane. The fact that the selectivity exists at low temperature, that it does not depend on cytoskeleton integrity and that there is a competition between the two aminophospholipids show that this translocation is not purely an endocytic process.

Animals↗
PubMed1986

Interaction of cytochrome c with mixed dimyristoylphosphatidylcholine-dimyristoylphosphatidylserine bilayers: a deuterium nuclear magnetic resonance study.

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of cytochrome c (from horse heart) with bilayers of mixed dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylserine (DMPS). Three types of labeled lipids were used: chain-perdeuterated phosphatidylcholine (DMPC-d54), chain-perdeuterated phosphatidylserine (DMPS-d54), and phosphatidylserine labeled at the alpha-position of the head group (DMPS-d2). Liposomes containing equimolar mixtures of DMPC and DMPS were found to bind cytochrome c with a maximum ratio of about 1 mg of cytochrome c per 1 mg of DMPS. The 2H NMR spectra of equimolar mixtures of DMPC-d54-DMPS and DMPC-DMPS-d54 were examined with and without cytochrome c. No change of the NMR spectra of either DMPC or DMPS could be detected after protein addition, for temperatures both above and below the phospholipid phase transition region. On the other hand, in the liquid-crystalline state, the transverse relaxation time, T2e, was reduced by 30-40% after protein addition. Measurements of the spin-lattice relaxation time, T1, showed, under all circumstances, multiple components. For simplicity, we have examined the shape of the relaxation curves at short and long times. Addition of protein increased by 2-fold the value of the slow T1 component of DMPS-d54 but not that of DMPC-d54. Partially relaxed spectroscopy allowed us to assign this slow component (at least in part) to the methyl group and C2H2 groups near the methyl end of the chains, i.e., far from the binding sites of the extrinsic protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme.

When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗
PubMed1986

[Energy-dependent translocation of amino-phospholipids in the erythrocyte membrane].

In this review, we show how the stability of the asymmetric transverse distribution of phospholipids and the physiological role of the asymmetric distribution can be explained. Experiments with paramagnetic or fluorescent lipids enabled us to show that in fresh red blood cells, i.e. containing ATP, and in resealed ghosts containing ATP (1 mM) the amino derivatives (phosphatidylserine and phosphatidylethanolamine) are selectively transported from the outer monolayer to the inner monolayer of the membranes. On the other hand, phosphatidylcholine and sphingomyelin are not carried and diffuse spontaneously with a very long characteristic time. The ATP-dependent carrier mechanism can be inhibited by protein reacting groups (N-ethyl maleimide and ortho-vanadate), which very probably implies a transmembrane protein specific for amino phospholipids. The affinity for phosphatidylserine seems slightly higher than that for phosphatidylethanolamine. In addition we show the close parallel between the transverse distribution of phospholipids and cell shape. This leads us to suggest that the phospholipid translocation would be used to maintain the natural discoid shape of red blood cells. A possible generalisation of this mechanism to other cells and its implications for endocytosis are discussed.

Adenosine Triphosphate↗
PubMed1986

Asymmetric lateral mobility of phospholipids in the human erythrocyte membrane.

The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified from corn. The lateral mobility of the fluorescent phospholipids was measured by using an extension of the classical photobleaching recovery technique that takes advantage of a modulated fringe pattern and provides a high sensitivity. In intact erythrocytes and in ghosts resealed in the presence of ATP, the fluorescence-contrast curves after photobleaching decayed biexponentially corresponding to two lateral diffusion constants. With NBD-phosphatidylcholine, the majority of the signal corresponded to a "slow" component (1.08 X 10(-9) cm2/sec at 20 degrees C), whereas with the amino derivatives the majority of the signal corresponded to a "fast" component (5.14 X 10(-9) cm2/sec at 20 degrees C). If the ghosts were resealed without ATP, the fast component of the aminophospholipids disappeared. We interpret these results as follows: (i) Provided the cells or the ghosts contain ATP, the three fluorescent phospholipids distribute spontaneously between inner and outer leaflets as endogenous phospholipids, namely NBD-phosphatidylcholine is located in the outer leaflet, while both aminophospholipids are preferentially located in the inner leaflet. (ii) The viscosity of the inner leaflet of human erythrocyte membranes is lower than that of the outer leaflet.

Adenosine Triphosphate↗
PubMed1985

Absence of transbilayer diffusion of spin-labeled sphingomyelin on human erythrocytes. Comparison with the diffusion of several spin-labeled glycerophospholipids.

We have measured the transbilayer diffusion at 4 degrees C of spin labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid in the human erythrocyte membrane. Measurements were also carried out in ghosts, released without ATP, and on large unilamellar vesicles made with total lipid extract. As reported previously (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), the amino phospholipids are rapidly transported from the outer to the inner leaflet on fresh erythrocytes, whereas phosphatidylcholine diffuses slowly. We now show that phosphatidic acid behaves like phosphatidylcholine: approximately 10% is internalized in 5 h at 4 degrees C. Under the same experimental conditions, no inward transport of sphingomyelin can be detected. In ghosts resealed without ATP, all glycerophospholipids tested diffuse slowly from the outer to the inner leaflet (approx. 10% in 5 h) while no transport of sphingomyelin is seen. Finally in lipid vesicles, the inward diffusion of all glycerophospholipids is less than 2% in 5 h and a very small transport of sphingomyelin can be measured. These results confirm the existence of a selective inward aminophospholipid transport of fresh erythrocytes and suggest a slow and passive diffusion of all phospholipids on ghosts, resealed without ATP, as well as on lipid vesicles.

Adenosine Triphosphate↗
PubMed1985

Strong interactions between a spin-labeled cholesterol analog and erythrocyte proteins in the human erythrocyte membrane.

We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost suspension. With intact erythrocytes or white ghosts in isotonic buffer, the ESR spectrum is a superposition of a mobile component and a strongly immobilized component (outer hyperfine splitting 61-63 G). The latter corresponds to approx. 45% of the signal, a percentage which is barely affected by varying the temperature between 5 and 37 degrees C. Removal of the cytoskeletal proteins spectrin and actin by low ionic strength treatment or of all extrinsic proteins by alkali treatment of ghosts reduces the immobilized fraction to approx. 25%. The effect of controlled proteolysis of intrinsic proteins was also tested. Pre-treatment of cells with chymotrypsin or pre-treatment of unsealed ghosts with trypsin has no effect on the ESR spectrum obtained with alkali-treated membranes. On the other hand, after chymotrypsin treatment of unsealed ghost, which reduces the band 3 protein to a 17.5 kDa membrane fragment, the strongly immobilized component is no longer observable. These data show that the cholesterol analog 26-nor-25-doxylcholestanol interacts strongly with one or several proteins of the erythrocyte membrane. That the intrinsic protein band 3 is involved is suggested by the disappearance of the immobilized fraction occurring upon chymotrypsin digestion of this protein. Our results are thus consistent with the proposal of a selective cholesterol-band 3 interaction in the erythrocyte membrane (Schubert, D. and Boss, K. (1982) FEBS Lett. 150, 4-8). Our data also suggest that this interaction is influenced by cytoskeletal proteins, an effect which can be explained considering the known linking of band 3 to the erythrocyte cytoskeleton via ankyrin. Experiments have also been carried out with 3-doxylandrostanol, a more commonly used cholesterol spin-label analog. With this spin label, at all temperatures investigated, we found it impossible to demonstrate unambiguously the existence of two spectral components. It is suggested that 26-nor-25-doxylcholestanol is a better reporter of cholesterol behavior in membranes.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

Abnormality of phospholipid transverse diffusion in sickle erythrocytes.

We have used spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to compare the transverse diffusion rates of lipids in normal and sickle erythrocytes. The beta-chain of the spin-labeled lipids was a short chain (five carbons) providing the spin labels with a relative water solubility, and hence permitting their rapid incorporation into cell membranes. The orientation of the labeled lipids in the membranes was assayed by selective chemical reduction of the nitroxide labels embedded in the outer leaflet. We have found that all three spin-labeled phospholipids are initially incorporated in the outer leaflet. Upon incubation at 4 degrees C the aminophospholipids, not the phosphatidylcholine, diffuse toward the inner leaflet within 3 h. The transverse diffusion rate of aminophospholipids is reduced by 41% (phosphatidylserine) and 14% (phosphatidylethanolamine) in homozygote sickle cells (SS) when compared with normal cells (AA) or heterozygote cells (AS or SC). At equilibrium the asymmetric distribution of spin-labeled phospholipids resulting from this selective diffusion is also reduced in SS cells when compared with AA, SC, or AS cells. This reduced asymmetry was not found in a reticulocyte-rich blood sample (hemoglobin A), indicating that the age of the cell cannot be responsible for this phenomenon. Moreover, because at low temperatures the sickling process does not occur, the observed perturbations in phospholipid organization reflect preexisting membrane abnormalities in sickle cells. Ghosts loaded with ATP give the same results. Varying the concentration of intracellular calcium had no effect on lipid diffusion, except at very high free calcium concentrations (3 microM) when diffusion was practically abolished. We suggest that membrane protein alterations may be part of the explanation of the observed abnormalities.

Anemia, Sickle Cell↗