Study of the transverse diffusion of spin labeled phospholipids in biological membranes. I. Human red bloods 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 beta acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22 degrees C with sonicated spin-labed 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 beta chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0 degrees C 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 creaging 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 degrees 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°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).     

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).     

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.     

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.     

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.     

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.     

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)     

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.     

Dynamic states of phospholipids in Escherichia coli B membrane. Electron spin resonance studies with biosynthetically generated phospholipid spin labels

Mobility of phospholipid hydrocarbons in the Escherichia coli B membrane fractions was studied by labeling phosphatidylethanolamine or phosphatidylglycerol in situ by biosynthetic incorporation of the spin label. For this purpose, CDP-diacylglycerol spin label was synthesized from phosphatidic acid spin label and cytidine 5'-phosphoromorpholidate and purified by thin-layer chromatography. DCP-diacylglycerol spin label was then incorporated into phospholipids biosynthetically. ESR spectra of these E. coli B membrane fractions showed that phosphatidylglycerol tended to interact with membrane proteins through the mediation of Mg2+, whereas phosphatidylethanolamine had less of this tendency and was more involved in the formation of the bulk of the bilayer continuum of the membrane. These conclusions were also supported by labeling membranes with exogenous spin-labeled phospholipids, although there was some indication that exogenous phospholipids were incorporated into sites different from the sites of incorporation of phospholipids newly synthesized in situ.     

Incorporation of spin-labeled ganglioside analogues into cell and liposomal membranes

Ganglioside analogues (gangliosides) with an electron spin resonance label in a long aliphatic hydrocarbon chain were used to investigate the possible insertion of the sialoglycolipid into the plasma membrane of cells. Three types of ESR signals observed in the labeled glycolipids were distinguished. They characteristically indicate an isotropic tumbling motion of spin label in solution, the micellar state of the glycolipid, and an anisotropic motion in a lipid bilayer. Below CMC, gangliosidoide carrying one aliphatic hydrocarbon chain showed an isotropic tumbling motion. After the gangliosidoide had been incubated with liposomes or blood cells, there was an immediate change to an ESR signal showing an anisotropic motion. The signal was typical of the spin-label in liposomes prepared in the presence of spin-labeled sialoglycolipid. It can be concluded that the gangliosidoide was inserted into the lipid phase of liposomal or cellular membranes from the incubation medium. The overall splitting (2A parallel) of 5SL-gangliosidoides in membranes was larger than those of 5SL-galactosylceramide, 5SL-phosphatidylcholine, and 5SL-stearic acid, though the 2A parallel of 12SL-gangliosidoide was almost the same as those of other lipids having a nitroxide group in the 12-position of an acyl chain. This indicates that the head group movement is restricted in gangliosidoide molecules.     

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.     

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.     

Free fatty acids and esters can be immobilized by receptor rich membranes from Torpedo marmorata but not phospholipid acyl chains.

A long chain spin labeled fatty acid and the corresponding ester have been introduced into receptor rich membranes from Torpedo Marmorata. Superimposed to a mobile component, typical of the lipid phase, a strongly immobilized component is seen on the ESR spectra, both at low temperature (?4°C) and at room temperature. An estimation of the amount of immobilized signal as a function of the concentration of spin label in the membrane shows that a saturation is reached which corresponds to approximately twice the concentration of receptor protein. In the same membranes, a spin labeled phosphatidylcholine was introduced by the release of the phosphatidylcholine analog from purified phosphatidylcholine exchange protein, preloaded with this spin label. No immobilized component is seen in this latter case even at low temperatures. Therefore the immobilized component seen with the fatty acid cannot be considered as reporting on an immobilized boundary layer of phospholipids surrounding the proteins. We attribute the immobilized signal seen with fatty acids and esters to a particular interaction of amphiphilic molecules with the cholinergic receptor protein. Very likely this effect can be associated with the local anaesthetic effect detected previously with this fatty acid.     

Transfer of phosphatidylcholine between different membranes in Tetrahymena as studied by spin labeling

Transfer of phosphatidylcholine molecules between different membrane fractions of Tetrahymena pyriformis cells grown at 15, 27 and 39.5°C was studied by electron spin resonance (ESR). Microsomes were labeled densely with a phosphatidylcholine spin label and the spin-labeled microsomes were incubated with non-labeled cilia, pellicles or microsomes. The transfer of the phosphatidylcholine spin labels was measured by decrease in the exchange broadening of the electron spin resonance spectrum. In one experiment, the lipid transfer was measured between ³²P-labeled microsomes and non-labeled pellicles by use of their radioactivity. The result was in good agreement with that by ESR. The fluidity of the membrane was estimated using a fatty-acid spin label incorporated into the membranes. Transfer between lipid vesicles was also studied. The results obtained were as follows: (1) The transfer between sonicated vesicles of egg- or dipalmitoyl phosphatidylcholine occurred rapidly in the liquid crystalline phase, with an activation energy of 20 kcal/mol, whereas it hardly occurred in the solid crystalline phase. (2) The transfer rate between microsomal membranes increased with temperature, and an activation energy of the reaction was 17.8 kcal/mol. (3) The transfer from the spin-labeled microsomes to subcellular membranes of the cells grown at 15°C was larger than that to the membranes of the cells grown at 39.5°C. The membrane fluidity was larger for the cells grown at lower temperature. (4) Similar tendency was observed for the transfer between microsomal lipid vesicles prepared from the cells grown at 15°C and at 39.5°C. (5) The transfer from microsomes to various membrane fractions increased in the order, cilia < pellicles < microsomes. The order of increase in the membrane fluidity was cilia < microsomes < pellicles, although the difference between microsomes and pellicles was slight. These results indicate a crucial role of the membrane fluidity in the transfer reaction. (6) Some evidence supported the idea that the lipid transfer between these organelles occurred through the lipid exchange rather than through the fusion.     

Studies of the mobility of maleimide spin labels within the erythrocyte membrane

We have confirmed a method yielding reproducible and reliable spectrometric parameters derived from spin-labeled erythrocyte ghosts using nitroxide derivatives of maleimide compounds. The disorder parameter, W/S, was shown to vary with changes in the structure of the label, the conditions utilized for labeling such as ionic strength and erythrocyte age and the presence of drugs such as alcohol and acetaminophen. The nitroxide spectrum was also found to change with increasing and decreasing temperature in an irreversible manner. These findings should permit increased reliance to be placed on the spin-labeling technique when used to monitor changes in membrane lipid or protein assembly.     

Labeling of bovine heart cytochrome c oxidase with analogues of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.     

Glycosphingolipids in membrane architecture

As part of a program to investigate the behavior and interactions of glycolipids in biological membranes we have synthesized spin-labeled derivatives of 2 families of carbohydrate-bearing ceramides (glycosphingolipids): simple neutral glycolipids and gangliosides. Galactosyl ceramide has been synthesized with the spin label at 3 different positions on the fatty acid chain. It has been studied in bilayers of various different lipids and lipid mixtures and compared to the corresponding phospholipid spin labels. Considerable similarity has been found between the behavior of galactosyl ceramide and phosphatidylcholine. These similarities include a negligible flip-flop rate, a flexibility gradient in the acyl chains, and exclusion from phosphatidylserine domains in the face of a Ca²⁺-induced lateral phase separation. Evidence for dramatic clustering of simple neutral glycolipids has not been found. Glycosphingolipids do seem to have the capacity to increase rigidity in fluid lipid bilayers. A general procedure has been developed for covalent attachment of a nitroxide spin label to the headgroup region of complex glycolipids such as gangliosides. Studies of beef brain gangliosides labeled in this manner and incorporated into bilayers of phosphatidylcholine indicate that the headgroup oligosaccharides are in rapid, random motion as opposed to being in any way immobilized. This headgroup mobility depends very little on the fluidity or rigidity of the bilayer. However, headgroup mobility decreases, perhaps as a result of cooperative headgroup interactions, with increasing bilayer concentration of unlabeled ganglioside.     

Transmembrane phospholipid motions induced by F glycoprotein in hemagglutinating virus of Japan.

Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.