Study of the transverse diffusion of spin labelled phospholipids in biological membranes. I. Human red blood cells

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the β acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22°C with sonicated spin-labeled phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the β chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0dgC allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creating a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37°C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.     

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: use of phosphatidylcholine exchange protein.

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated "inner membrane+matrix" particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22 degrees C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidycholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at 0 degrees C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incorporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0 degrees C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25 degrees C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).     

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: Use of phosphatidylcholine exchange protein

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated “inner membrane+matrix” particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22°C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group.When spin-labeled phosphatidylcholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at O°C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incoporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0°C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling.The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25°C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis.

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe3+, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 degrees C, while that for phosphatidylserine spin label had only one transition at 30 degrees C. When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogenous fluidity. Mg2+ or Mg2+ + ATP prevented the hemolysis-induced spectral changed. Ca2+ did not prevent the homogenization and acted antagonistically to Mg2+. The heterogeneity preservation by Mg2+ was nullified by trypsin, pronase or N-ethylmaleimide added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg2+ was dependent on hemolysis temperature, starting to decrease at 18 degrees C and vanishing at 40 degrees C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Effect of benzyl alcohol on phospholipid transverse mobility in human erythrocyte membrane.

The effect of benzyl alcohol on the transverse mobility and repartition of phospholipids in the human erythrocyte membrane was investigated using electron spin resonance and morphological modification of red blood cells. Transmembrane internalization rates and equilibrium distribution in red blood cells of short-chain spin-labeled phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine were strongly modified by treatment with 10-70 mM benzyl alcohol. A dual effect was observed: (a) at 4 degrees C and 37 degrees C there was an N-ethylmaleimide-sensitive, long lasting and fully reversible increase in the spin-labeled phosphatidylserine and phosphatidylethanolamine internalization rate; (b) at 37 degrees C, an enhancement of N-ethylmaleimide-insensitive fluxes of all the labeled phospholipids through the membrane occurred. Both effects were dose-dependent. Erythrocytes submitted to benzyl alcohol incubation also showed dose-dependent shape changes: an immediate one from discocytes to echinocytes, followed by a slower N-ethylmaleimide- and ATP-dependent change to stomatocytes. Moreover, benzyl alcohol treatment was shown to lead to enhanced hydrolysis of intracellular ATP. All the effects of benzyl alcohol can be described as an accumulation of labeled phosphatidylethanolamine (and labeled phosphatidylcholine at 37 degrees C) in the inner leaflet. This can be interpreted as a perturbation of the erythrocyte membrane, leading to an energy-consuming specific increase in aminophospholipid translocase activity, in addition to a slow and passive bidirectional flux of all phospholipids at 37 degrees C.     

Micellar formation of spin-labeled fatty acyl derivatives of lipophilic muramyl dipeptides and their incorporation into liposomal membranes

A lipophilic muramyl dipeptide (MDP) with a nitroxide moiety in its acyl chain (SL-MDP) and its N-methyl derivative (SL-methyl MDP) were synthesized. The SL-MDPs formed micelles (cmc, 0.1-0.3 mM). The ESR spectra of the SL-MDPs in phosphatidylcholine (PC) liposomes at 25 degrees C consisted of an anisotropic signal and three sharp lines, indicating that both SL-MDPs partitioned between membranes and aqueous phase. The amounts of the SL-MDPs in membranes depended on the phospholipid species and the cholesterol (Chol) content, but no appreciable difference was observed between SL-MDPs. The SL-MDPs partitioned well at 25 degrees C into egg yolk PC liposomes but not into pure dipalmitoylphosphatidylcholine (DPPC), suggesting that the incorporation may be related to the membrane fluidity. Chol enhanced the incorporation into both phospholipids. The mobilities of the SL-MDPs in the membranes were less than that of the corresponding spin-labeled fatty acid. Comparison of the mobilities among SL-MDPs, spin-labeled ganglioside and spin-labeled galactosylceramide showed that the hydrophilicity of the polar group may influence the immobilization of their acyl chains.     

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)     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

Protein-induced vertical lipid dislocation in a model membrane system: spin-label relaxation studies on avidin-biotinylphosphatidylethanolamine interactions.

The change in vertical location of spin-labeled N-biotinyl phosphatidylethanolamine in fluid-phase dimyristoyl phosphatidylcholine bilayer membranes, on binding avidin to the biotinyl headgroup, has been investigated by progressive saturation electron spin resonance measurements. Spin-labeled phospholipids were present at a concentration of 1 mol%, relative to total membrane lipids. For avidin-bound N-biotinyl phosphatidylethanolamine spin-labeled on the 8 C atom of the sn-2 chain, the relaxation enhancement induced by 30 mM Ni2+ ions confined to the aqueous phase was 2.5 times that induced by saturating molecular oxygen, which is preferentially concentrated in the hydrophobic core of the membrane. For phosphatidylcholine also spin-labeled at the 8 position of the sn-2 chain, this ratio was reversed: the relaxation enhancement by Ni2+ ions was half that induced by molecular oxygen. In the absence of avidin, the enhancement by either relaxant was the same for both spin-labeled phospholipids. For a double-labeled system, in which both N-biotinyl phosphatidylethanolamine and phosphatidylcholine were spin-labeled on the 12 C atom of the sn-2 chain, the relaxation rate in the absence of avidin was greater than that predicted from linear additivity of the corresponding singly labeled systems, because of mutual spin-spin interactions between the two labeled lipid species. On binding of avidin to the N-biotinyl phosphatidylethanolamine, this relaxation enhancement by mutual spin-spin interaction was very much decreased. These results indicate that, on binding of avidin to the lipid headgroup, N-biotinyl phosphatidylethanolamine is lifted vertically within the membrane, relative to the phosphatidylcholine host lipids. The specific binding of avidin to N-biotinyl phosphatidylethanolamine parallels the liftase activity proposed for activator proteins associated with the action of certain gangliosidases.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe³⁺, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg²⁺ or $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ prevented the hemolysis-induced spectral changes. Ca²⁺ did not prevent the homogenization and acted antagonistically to Mg²⁺. The heterogeneity preservation by Mg²⁺ was nullified by trypsin, pronase or $\text{N-}\text{ethylmaleimide}$ added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg²⁺ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

Spin-label electron spin resonance study of bacteriophage M13 coat protein incorporation into mixed lipid bilayers

The major coat protein of bacteriophage M13 was incorporated in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (80/20 w/w) vesicles probed with different spin-labeled phospholipids, labeled on the C-14 atom of the sn-2 chain. The specificity for a series of phospholipids was determined from a motionally restricted component seen in the electron spin resonance (ESR) spectra of vesicles with the coat protein incorporated. At 30 degrees C and pH 8, the fraction of motionally restricted phosphatidic acid spin-label is 0.36, 0.52, and 0.72 for lipid/protein ratios of 18, 14, and 9 mol/mol, respectively. The ESR spectra, analyzed by digital subtraction, resulted in a phospholipid preference following the pattern cardiolipin = phosphatidic acid greater than stearic acid = phosphatidylserine = phosphatidylglycerol greater than phosphatidylcholine = phosphatidylethanolamine. The specificities found are related to the composition of the target Escherichia coli cytoplasmic membrane.     

Effect of fatty acyl chain length of phosphatidylcholine on their transfer from liposomes to erythrocytes and transverse diffusion in the membranes inferred by TEMPO-phosphatidylcholine spin probes

TEMPO-phosphatidylcholine (PC) spin probes which have homologous saturated acyl chains of 10, 12, 14 and 16 carbon atoms, were synthesized as analogues of PC. Transfer of TEMPO-PCs from liposomal membrane to the ghost membrane of human erythrocyte and transverse diffusion of TEMPO-PCs within the membrane of intact erythrocytes were determined by measurement of spontaneous increase and decrease in signal amplitude of an anisotropic triplet spectrum, due to dilution of the label by natural phospholipid of the membrane and reduction of the label by the cytoplasmic content of the erythrocyte, respectively. TEMPO-PC molecules in TEMPO-PC liposomes, except dipalmitoyl TEMPO-PC, were rapidly incorporated into the ghost membrane by incubation at 37 degrees C; the PC having shorter acyl chains was transferred faster. The cytoplasmic content of the erythrocyte rapidly reduced the nitroxide radical of the spin probe. The central peak height of ESR signal was once increased by incorporation of TEMPO-PC into the erythrocyte membrane and then was spontaneously decreased during further incubation at 37 degrees C. This decrease indicates that PC molecules traverse from the outer to the inner layer of the membrane lipid bilayer. The decrease of signal amplitude was faster with PC of shorter acyl chain. These findings suggest that both transfer between membranes and transverse diffusion in the membrane may be favored to the PC species with shorter acyl chains.     

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.     

Membranotropic effect of phosphocreatine and its structural analogs]

The effects of phosphocreatine (PCr) and its analogues (creatine, phosphocreatinine, phosphoarginine and inorganic phosphate) on liposomal and erythrocyte membranes and on the sarcolemmal membrane of cardiomyocytes were studied. The ESR spectrum of the spin-labeled probe, 5-doxyl-stearate, incorporated into the membrane were recorded for analysis of the structural order of the phospholipid bilayer of these membranes. PCr and its analogues had no effect on the structure of the phospholipid bilayer in liposomes; this effect was temperature-independent. However, in erythrocyte and sarcolemmal membranes the rigidity of the membranes was increased by these compounds (except for creatine) at temperatures above 38-40 degrees C. Analysis of these and literary data revealed that cardiac cell membranes may be the site of protective action of PCr on the ischemic myocardium. The lack of effect on liposomes may suggest that the membrane-stabilizing effect of PCr depends on the presence of membrane proteins. The compounds under study may influence the lipid-protein interactions by increasing the rigidity of membrane phospholipids. These membranotropic effects may be due to the interaction of charged molecules of the compounds with polar heads of phospholipids and/or polar groups of proteins in the membrane interphase which, in turn, may influence the packing of hydrophobic fatty acid chains.     

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+.     

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.     

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)     

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.     

Rotational relaxation times of 1,6-diphenyl-1,3,5-hexatriene in phospholipids isolated from LM cell membranes. Effects of phospholipid polar head-group and fatty acid composition.

Phospholipids were isolated from mitochondrial, microsomal, and plasma membranes of LM cells and fractionated into individual phospholipid classes on silicic acid columns. The fatty acid composition and the rotational relaxation time of 1,6-diphenyl-1,3,5-hexatriene (DPH) were determined for each phospholipid class. Sphingomyelin was the only phospholipid isolated from LM cell membranes that showed a phase transition within the temperature range investigated, 5-40 degrees C. The rotational relaxation times for DPH were lowest in phosphatidylcholine in all the membrane fractions. Phosphatidylcholine isolated from the three membrane fractions of choline-supplemented cells had similar rotational relaxation times and phosphatidylcholine isolated from microsomal membranes of linoleate-supplemented cells had lower rotational relaxation times. The results indicate that the differences in the rotational relaxation times of DPH between mitochondrial, microsomal, and plasma membrane phospholipids could be explained primarily by differences in the polar head-group composition, while differences in the fatty acid composition had only a minor effect. This provides a basis for understanding how the different lipid components in these cells contribute to membrane fluidity.     

Formation of omega- and (omega-1)-hydroxyfatty acids and plausible formation of ketofatty acid from microsomal phospholipids

Rat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C]phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60 min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C]phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.