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

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

The distribution of erythrocyte phospholipids in hereditary spherocytosis demonstrates a minimal role for erythrocyte spectrin on phospholipid diffusion and asymmetry

In the human erythrocyte membrane phosphatidylcholine and sphingomyelin reside mainly in the outer leaflet, whereas the aminophospholipids, phosphatidylethanolamine and phosphatidylserine, are mainly found in the inner leaflet. Maintenance of phospholipid asymmetry has been assumed to involve interactions between the aminophospholipids and the membrane skeleton, in particular spectrin. To investigate whether spectrin contributes to maintaining the phospholipid transbilayer distribution and kinetics of redistribution, we studied erythrocytes from hereditary spherocytosis patients whose spectrin levels ranged from 34% to 82% of normal. The phospholipid composition and the accessibility of membrane phospholipids to hydrolysis by phospholipases were in the normal range. Spin-labeled phosphatidylserine and phosphatidylethanolamine analogues that had been introduced into the outer leaflet were rapidly transported at 37 degrees C to the inner leaflet, whereas the redistribution of spin-labeled phosphatidylcholine was slower. The kinetics of transbilayer movement of these spin-labeled phospholipid in all samples was in the normal range and was not affected by the level of spectrin. Although these erythrocyte membranes contained as little as 34% of the normal level of spectrin and were characterized by several physical abnormalities, the composition, distribution, and transbilayer kinetics of the phospholipids were found to be normal. We therefore conclude that spectrin plays, at best, only a minor role in maintaining the distribution of erythrocyte membrane phospholipid.     

Identification of a functional role for lipid asymmetry in biological membranes: Phosphatidylserine-skeletal protein interactions modulate membrane stability

Asymmetric distribution of phospholipids is ubiquitous in the plasma membranes of many eukaryotic cells. The majority of the aminophospholipids are located in the inner leaflet whereas the cholinephospholipids are localized predominantly in the outer leaflet. Several functional roles for asymmetric phospholipid distribution in plasma membranes have been suggested. Disruption of lipid asymmetry creates a procoagulant surface on platelets and serves as a trigger for macrophage recognition of apoptotic cells. Furthermore, the dynamic process of phospholipid translocation regulates important cellular events such as membrane budding and endocytosis. In the present study, we used the red cell membrane as the model system to explore the contribution of phospholipid asymmetry to the maintenance of membrane mechanical properties. We prepared two different types of membranes in terms of their phospholipid distribution, one in which phospholipids were scrambled and the other in which the asymmetric distribution of phospholipids was maintained and quantitated their mechanical properties. We documented that maintenance of asymmetric distribution of phospholipids resulted in improved membrane mechanical stability. The greater difficulty in extracting the spectrin-actin complex at low-ionic strength from the membranes with asymmetric phospholipid distribution further suggested the involvement of interactions between aminophospholipids in the inner leaflet and skeletal proteins in modulating mechanical stability of the red cell membrane. These findings have enabled us to document a functional role of lipid asymmetry in regulating membrane material properties.     

beta-Adrenergic receptor agonists increase phospholipid methylation, membrane fluidity, and beta-adrenergic receptor-adenylate cyclase coupling.

The beta-adrenergic agonist L-isoproterenol stimulated the enzymic synthesis of phosphatidyl-N-monomethylethanolamine and phosphatidylcholine in rat reticulocyte ghosts containing the methyl donor S-adenosyl-L-methionine. The stimulation was stereospecific, dose-dependent, and inhibited by the beta-adrenergic agonist propranolol. The addition of GTP inside the resealed ghosts shifted the dose-response of phospholipid methylation by L-isoproterenol to the left by 2 orders of magnitude. Direct stimulation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with sodium fluoride or cholera toxin did not increase the methylation of phospholipids. At a concentration of S-adenosyl-L-methionine that stimulates synthesis of phosphatidyl-N-monomethylethanolamine, the activity of isoproterenol-sensitive adenylate cyclase was increased 2-fold without changes in the basal activity of adenylate cyclase and the number of beta-adrenergic receptors. The increase of phospholipid methylation by L-isoproterenol decreased membrane viscosity and increased translocation of methylated lipids. These findings indicate that enhancement of phospholipid methylation by L-isoproterenol decreases membrane microviscosity and thus increases lateral movement of the beta-adrenergic receptors and coupling with adenylate cyclase.     

Prolonged storage of red blood cells affects aminophospholipid translocase activity

BACKGROUND AND OBJECTIVES: Loss of phospholipid asymmetry in the membrane of red blood cells (RBC) results in exposure of phosphatidylserine (PS) and to subsequent removal from the circulation. In this study, we investigated the effect of long-term storage of RBCs on two activities affecting phospholipid asymmetry: the ATP-dependent aminophospholipid translocase (or flippase, transporting PS from the outer to the inner leaflet) and phospholipid scrambling (which will move PS from the inner to the outer leaflet). MATERIALS AND METHODS: Standard leukodepleted RBC concentrates were stored in saline-adenine-glucose-mannitol (SAGM) at 4 degrees C for up to 7 weeks. PS exposure was determined by measurement of AnnexinV-FITC binding to the cells, flippase activity by measurement of the inward translocation of NBD-labelled PS. Scrambling activity was determined by following the inward translocation of fluorescent NBD-phosphatidylcholine. In parallel, intracellular ATP levels were determined. RESULTS: PS exposure amounted to only 1.5 +/- 0.3% positive cells (n = 8) after 5 weeks of storage, which slightly increased to 3.5 +/- 0.7% (n = 8) after 7 weeks of storage. Flippase activity started to decrease after 21 days of storage and reached 81 +/- 5% of the control value after 5 weeks of storage (n = 6) and 59 +/- 6% (n = 6) after 7 weeks. Also in RBC obtained by apheresis, flippase activity decreased upon storage. Scrambling activity remained virtually absent during storage, explaining the low PS exposure despite the decrease in flippase activity. Rejuvenation of RBC after 7 weeks to increase ATP levels only partially restored flippase activity, but in combination with a correction of the intracellular pH to that of fresh cells, almost complete restoration was achieved. The decrease in flippase activity after prolonged storage did make the RBCs more prone to PS exposure after activation of phospholipid scrambling. CONCLUSION: This study shows that, although PS exposure remains low, prolonged storage does compromise the RBC membrane by affecting flippase activity. When the metabolic changes induced by storage are corrected, flippase activity can be restored.     

A unique phospholipid organization in bovine erythrocyte membranes

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.     

Phospholipid transmembrane domains and lateral diffusion in fibroblasts.

The lateral diffusion of fluorescent phospholipids in cultured Chinese hamster lung fibroblasts was examined by modulated fringe pattern photobleaching. When cells were labeled and maintained at 7 degrees C, the fluorescence remained localized at the plasma membrane. N-[6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] sphingosylphosphocholine (C6-NBD-SphPCho) and 1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylcholine (C6-NBD-PtdCho) both diffused with the same apparent lateral diffusion coefficient (D1 approximately 0.3 x 10(-9) cm2/s). By contrast, the phosphatidylserine derivative (1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylserine (C6-NBD-Ptd-Ser)) gave rise to two diffusional components: a slow component, D1, analogous to that measured with the choline-containing lipids, and a fast component (D2 approximately 2 x 10(-9) cm2/s). The fast component only exists in ATP-containing cells. It was shown to be associated with C6-NBD-PtdSer translocated to the inner leaflet. This indicates that the two leaflets form very different membranous domains. At higher temperature, the same difference in mobility was observed between the choline-containing lipids and the aminolipid. However, with C6-NBD-SphPCho, a fraction of very slowly diffusing or quasi-immobilized probes gradually appeared with time. This could be attributed to sphingomyelin located in small organelles after internalization. From the amplitude of this component registered at different intervals, we calculated that approximately 50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP- and microtubule-dependent process.     

Phosphatidylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface.

Liposomes formulated to resemble the outer leaflet of the erythrocyte membrane were found to substantially avoid recognition and clearance by the reticuloendothelial system. When these models of the erythrocyte surface were modified by the incorporation of greater than 2 mol % of phosphatidylserine (PtdSer), their ability to remain in the circulation of mice was greatly reduced. To examine whether this altered behavior was the consequence of an alteration in bilayer organization induced by PtdSer, a method utilizing the fluorescent dye merocyanine 540 was used to assess the packing of external phospholipids. No significant difference in overall membrane lipid organization was detected between liposomes containing 2 or 3 mol % of PtdSer, at which dramatic differences in recognition and clearance occurred. These results exclude alterations in phospholipid packing as an indirect cause of increased clearance of PtdSer-containing liposomes and implicate PtdSer directly in recognition by the reticuloendothelial system.     

Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine.

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09-0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-[N-[6(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino]caproyl]-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to approximately 40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.     

Sickled erythrocytes accelerate clotting in vitro: an effect of abnormal membrane lipid asymmetry

A membrane lipid abnormality induced by sickling and found as a permanent alteration in the irreversibly sickled cell (ISC) is the rearrangement of phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS) from the inner to the exterior side of the lipid bilayer. Since PS can provide a catalytic surface for the binding of blood coagulation factors and thus can exhibit procoagulant activity, we investigated the influence of oxy and deoxy reversibly sickled cells (RSC) ass well as ISC on clotting in vitro. Red blood cells (RBC), as the source of phospholipid, were added to platelet-poor citrated plasma containing Russell's viper venom (RVV) and clotting time was measured after recalcification. The clotting time after addition of normal RBC and oxy- RSC was similar to the saline blank (100 sec). In contrast, both oxy- ISC and deoxy completely sickled RSC shortened clotting time by 30%. Using liposomes prepared with identical phospholipid composition to the outer lipid leaflet of either normal RBC, RSC or ISC clotting times similar to those with intact cells were achieved. Since the liposomes did not contain protein, accentuation of clotting appears to be related to abnormal phospholipid organization, in particular to the abnormal exposure to aminophospholipids on the outer surface of the membrane. This abnormality may contribute to the pathogenesis of the vaso- occlusive episode in sickle cell anemia.     

Transmembrane mobility of phospholipids in sickle erythrocytes: effect of deoxygenation on diffusion and asymmetry

We studied the effect of sickling on the transmembrane reorientation and distribution of phospholipids in the red blood cells of patients homozygous for sickle cell anemia (SS). To this purpose, we followed the redistribution kinetics of trace amounts of spin-labeled analogues of natural phospholipids first introduced in the membrane outer leaflet of normal or sickle erythrocytes exposed to air or nitrogen. Deoxygenation had no effect on the lipid redistribution kinetics in normal (AA) cell membranes. At atmospheric pO2, unfractionated SS cells were not different from normal cells. However, on deoxygenation inducing sickling, phosphatidylcholine passive diffusion was accelerated and the rate of the adenosine triphosphate-dependent transport of aminophospholipids was reduced, especially for phosphatidylserine. The stationary distribution of the aminophospholipids between the two leaflets was slightly less asymmetric, a phenomenon more pronounced with phosphatidylethanolamine. These changes were rapidly reversible on reoxygenation. When SS cells were separated by density, both dense and light cells exhibited the properties cited above. However, dense cells exposed to air possessed a lower aminophospholipid transport rate. These data favor the relationship between aminophospholipid translocase activity and phospholipid transmembrane asymmetry. Sickle cell disease is the first case of aminophospholipid translocase pathology.     

Reconstitution of ATP-dependent aminophospholipid translocation in proteoliposomes.

In addition to ion-pumping ATPases, most plasma membranes of animal cells contain a Mg2+ ATPase activity, the function of which is unknown. This enzyme, of apparent molecular mass 110 kDa, was purified from human erythrocyte membranes by a series of column chromatographic procedures after solubilization in Triton X-100. When reincorporated into artificial bilayers formed from phosphatidylcholine, it was able to transport a spin-labeled phosphatidylserine analogue from the inner to the outer membrane leaflet provided Mg2+ ATP was present in the incubation mixture. The ATP-dependent transport of the phosphatidylethanolamine analogue required the presence of an anionic phospholipid (e.g., phosphatidylinositol) in the outer membrane leaflet. In contrast the transmembrane distribution of spin-labeled phosphatidylcholine was unaffected in the same experimental conditions. This transmembrane movement of aminophospholipid analogues was inhibited by treatment of the proteoliposomes with a sulfhydryl reagent. We conclude that the Mg2+ ATPase is sufficient for the biochemical expression of the aminophospholipid translocase activity, which is responsible for the inward transport of phosphatidylserine and phosphatidylethanolamine within the erythrocyte membrane. The presence of this transport activity in many animal cell plasma membranes provides a function for the Mg2+ ATPase borne by these membranes.     

Drs2p-coupled aminophospholipid translocase activity in yeast Golgi membranes and relationship to in vivo function

Aminophospholipid translocases (APLTs) are defined primarily by their ability to flip fluorescent or spin-labeled derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external leaflet of a membrane bilayer to the cytosolic leaflet and are thought to establish phospholipid asymmetry in biological membranes. The identities of APLTs remain unknown, although candidate proteins include the Drs2p/ATPase II subfamily of P-type ATPases. Drs2p from budding yeast localizes to the trans-Golgi network (TGN), and here we show that this membrane contains an ATP-dependent APLT that flips 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) PS and PE derivatives from the luminal to the cytosolic leaflet. To assess the contribution of Drs2p to this activity, TGN membranes were prepared from strains harboring WT or temperature-sensitive alleles of DRS2 and null alleles of three other potential APLT genes (DNF1, DNF2, and DNF3). Assay of these membranes indicated that Drs2p was required for the ATP-dependent translocation of NBD-PS, whereas no active translocation of NBD-PE or NBD-phosphatidylcholine was detected. The specificity of Drs2p for NBD-PS suggested that translocation of PS would be required for the function of Drs2p in protein transport from the TGN. However, cho1 yeast strains that are unable to synthesize PS do not phenocopy drs2 but instead transport proteins normally via the secretory pathway. In addition, a drs2 cho1 double mutant retains drs2 transport defects. Therefore, whereas NBD-PS is a preferred substrate for Drs2p in vitro, endogenous PS is not an obligatory substrate in vivo for the role Drs2p plays in protein transport.     

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P4-ATPase ATP8A2

ATP8A2 is a P(4)-ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P(4)-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp(416)) in the P-type ATPase signature sequence and exists in E(1)P and E(2)P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp(196), Glu(198), Lys(873), and Asn(874) in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E(2)P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K(+) activation of dephosphorylation in Na(+),K(+)-ATPase. Lys(873) mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys(873), located in transmembrane segment M5 at a "hot spot" for cation binding in Ca(2+)-ATPase and Na(+),K(+)-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys(865) is unimportant for aminophospholipid sensitivity. Binding of Na(+), H(+), K(+), Cl(-), or Ca(2+) to the E(1) form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.     

Characterization of lipid domains in erythrocyte membranes.

Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species.     

Determination of molecular motion in membranes using periodic pattern photobleaching.

The lateral diffusion of a fluorescent phospholipid probe in oriented multibilayers of dimyristoylphosphatidylcholine has been measured by observing the redistribution of fluorescence after photobleaching of the membranes in a periodic pattern of parallel stripes. The diffusion constant D of the fluorescent lipid was found to vary between 1.5 X 10(-11) cm2/sec at 9.6 degrees and 2.0 X 10(-10) at 22.5 degrees in the monoclinic phase. Preliminary studies of dipalmitoylphosphatidylcholine liposomes in the Lbeta'' and Pbeta'' phases yielded diffusion constants of the order of 10(-11) cm2/sec. These data are relevant to earlier discussions of the rate of complement activation by hapten-sensitized liposomal membranes [Brûlet, P. and McConnell, H. M. (1976) Proc. Natl. Acad. Sci. USA 73,2977--2981; Parce, J. W., Henry, N. and McConnell, H.M. (1978) Proc. Natl. Acad. Sci. USA 75, 1515--1518)]. We have also used this method to study the motion of fluorescent antibodies bound to murine EL-4 tumor cells. Pattern photobleaching techniques have the advantages that cellular or liposomal translation has no major adverse effect on the measurements, that certain nondiffusive motions can be detected and characterized, and that diffusive or other motions can be recorded photographically.     

Membrane ultrastructural changes during calcium phosphate-induced fusion of human erythrocyte ghosts.

Nascent calcium phosphate promotes the agglutination and fusion of human erythrocyte ghosts. Membrane phospholipids of erythrocyte ghosts treated with Ca2+ and phosphate ions become exposed to attack by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) (Bacillus cereus). Freeze-fracture pictures of fused erythrocyte ghosts show the presence of regions deficient in intramemebrane particles in the protoplasmic face which we believe to be regions of fusion. Discontinuous regions of the protoplasmic and exoplasmic faces are observed, which are apparently intermediate stages in the process of fusion. TH-in-section electron micrographs reveal deposits of calcium phosphate in areas of contact and fusion of ghosts. Ca2+ in the presence of N-[tris(hydroxymethyl)methyl]glycine (Tricine) buffer causes the formation of blebs in the membrane but does not cause changes in the intramembrane particle pattern or induce fusion. It is suggested that nascent calcium phosphate acts by forming protein-free regions of phospholipid bilayer which can fuse readily.     

Lectin Binding and Perturbation of the Outer Surface of the Cell Membrane Induces a Transmembrane Organizational Alteration at the Inner Surface

Binding of Ricinus communis I agglutinin to the outer surface of resealed human erythrocyte ghosts results in an organizational perturbation that is translated to the inner membrane surface. The organizational change was detected by an enhancement in the chemical cross-linking of several erythrocyte membrane components by the bifunctional reagent, dimethyl malonimidate, resulting in their loss or reduction after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized erythrocyte components. Of the components that failed to appear, or are reduced in amount, on the gels (protein bands Ia, Ib, IVa, and VII), two are known to be the subunits of spectrin (bands Ia and Ib), an inner-surface peripheral protein. A new band, which was identified as R. communis lectin, appeared on the polyacrylamide gels of lectin-treated ghosts with or without crosslinking. The loss of spectrin and other bands after lectin treatment and chemical crosslinking was due to a specific transmembrane event because: (a) β-lactose, an inhibitor of R. communis agglutinin, prevented labeling of ghosts by the lectin and loss of spectrin and other erythrocyte components on gels after crosslinking; (b) use of inactive bifunctional or active monofunctional crosslinking reagents did not result in loss of spectrin or other components from lectin-treated ghosts; (c) the loss of spectrin and other components after lectin treatment and crosslinking was sensitive to temperature and lectin concentration; (d) no new bands appeared on the gels except for the band identified as R. communis agglutinin; (e) R. communis agglutinin does not interact with purified spectrin; and (f) previously published data indicate the R. communis lectin binds exclusively to the outer membrane surface while spectrin is located on the inner membrane surface. Perturbation of components of the outer membrane surface that can be translated to the cell interior by transmembrane linkages may provide a structural means of membrane communication that could be important in a variety of cellular control processes.     

pH gradients across phospholipid membranes caused by fast flip-flop of un-ionized fatty acids.

A central, unresolved question in cell physiology is how fatty acids move across cell membranes and whether protein(s) are required to facilitate transbilayer movement. We have developed a method for monitoring movement of fatty acids across protein-free model membranes (phospholipid bilayers). Pyranin, a water-soluble, pH-sensitive fluorescent molecule, was trapped inside well-sealed phosphatidylcholine vesicles (with or without cholesterol) in Hepes buffer (pH 7.4). Upon addition of a long-chain fatty acid (e.g., oleic acid) to the external buffer (also Hepes, pH 7.4), a decrease in fluorescence of pyranin was observed immediately (within 10 sec). This acidification of the internal volume was the result of the "flip" of un-ionized fatty acids to the inner leaflet, followed by a release of protons from approximately 50% of these fatty acid molecules (apparent pKa in the bilayer = 7.6). The proton gradient thus generated dissipated slowly because of slow cyclic proton transfer by fatty acids. Addition of bovine serum albumin to vesicles with fatty acids instantly removed the pH gradient, indicating complete removal of fatty acids, which requires rapid "flop" of fatty acids from the inner to the outer monolayer layer. Using a four-state kinetic diagram of fatty acids in membranes, we conclude that un-ionized fatty acid flip-flops rapidly (t1/2 < or = 2 sec) whereas ionized fatty acid flip-flops slowly (t1/2 of minutes). Since fatty acids move across phosphatidylcholine bilayers spontaneously and rapidly, complex mechanisms (e.g., transport proteins) may not be required for translocation of fatty acids in biological membranes. The proton movement accompanying fatty acid flip-flop is an important consideration for fatty acid metabolism in normal physiology and in disease states such as cardiac ischemia.     

Modulation of membrane protein lateral mobility by polyphosphates and polyamines

The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of “fluorescence redistribution after fusion.” Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.