The rapid transmembrane movement of cholesterol in small unilamellar vesicles.

The exchange of cholesterol between two populations of small unilamellar vesicles has been investigated using a new system. Uniformly sized egg lecithin-cholesterol vesicles containing [3H]cholesterol and the glycolipid N-palmitoyl-DL-dihydrolactocerebroside were used as donors, whereas similar vesicles containing unlabelled cholesterol and no glycolipid were used as cholesterol acceptors. The two populations of vesicles were separated with the castor bean lectin Ricinus communis. It was found that greater than 90% of the cholesterol in the donor vesicle could be exchanged with a single time constant, the half-time for the completion of this exchange process being 1.5 h at 37 degrees C. Therefore, the rate of transmembrane movement or flip-flop of cholesterol in these vesicles must be at least as fast as the intermembrane exchange process. Similar results were obtained using hemoglobin-free human erythrocyte ghosts as the acceptor membrane. If the molecular-sieve chromatography step used to fractionate the vesicles was omitted, a non-exchangeable pool of cholesterol was detected which was shown not to be due to the presence of multilamellar vesicles.     

Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exhange protein. Measurement of inside--outside transitions.

Purified phosphatidylcholine exchange protein was used to exchange phosphatidylcholine between homogeneous single-walled phosphatidylcholine vesicles and human erythrocyte ghosts. When excess ghosts were present, it was found that only 70% of the vesicle phosphatidylcholine was available for exchange. This fraction corresponds closely to the amount of phosphatidycholine in the outer monolayer of these vesicles, indicating that only the outer surface of the vesicle is accessible to the exchange protein. Also, it was found that all phosphatidylcholine introduced into vesicles by the exchange protein was available for subsequent exchange. Using the exchange protein, asymmetrical vesicles were prepared in which the outer monolayer was either enriched or depleted in radioactive phosphatidylcholine as compared to the inner monolayer. Re-equilibration of the radioactivity between the two surfaces of the vesicle (flip-flop) could not be detected, even after 5 days at 37degrees. It is estimated that the half-time for flip-flop is in excess of 11 days at 37degrees. These results indicate that the properties of the exchange protein can be expolited to measure phosphatidylcholine flip-flop rates and possible phosphatidylcholine asymmetry in biological and model membranes, without altering the structure of the membrane.     

Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts.

Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.     

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

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

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.     

Flip-Flop-Induced Relaxation of Bending Energy: Implications for Membrane Remodeling

Cellular and organellar membranes are dynamic materials that underlie many aspects of cell biology. Biological membranes have long been thought of as elastic materials with respect to bending deformations. A wealth of theory and experimentation on pure phospholipid membranes provides abundant support for this idea. However, biological membranes are not composed solely of phospholipids—they also incorporate a variety of amphiphilic molecules that undergo rapid transbilayer flip-flop. Here we describe several experimental systems that demonstrate deformation-induced molecular flip-flop. First we use a fluorescence assay to track osmotically controlled membrane deformation in single component fatty acid vesicles, and show that the relaxation of the induced bending stress is mediated by fatty acid flip-flop. We then look at two-component phospholipid/cholesterol composite vesicles. We use NMR to show that the steady-state rate of interleaflet diffusion of cholesterol is fast relative to biological membrane remodeling. We then use a Förster resonance energy transfer assay to detect the transbilayer movement of cholesterol upon deformation. We suggest that our results can be interpreted by modifying the area difference elasticity model to account for the time-dependent relaxation of bending energy. Our findings suggest that rapid interleaflet diffusion of cholesterol may play a role in membrane remodeling in vivo. We suggest that the molecular characteristics of sterols make them evolutionarily preferred mediators of stress relaxation, and that the universal presence of sterols in the membranes of eukaryotes, even at low concentrations, reflects the importance of membrane remodeling in eukaryotic cells.     

Movement of cholesterol between vesicles prepared with different phospholipids or sizes

The rates of exchange of [4-14C]cholesterol between lipid vesicles prepared with different phospholipids and with different sizes have been measured. The first-order rate constants were higher using vesicles prepared from phosphatidylcholines with highly branched or polyunsaturated fatty acyl chains than with saturated diacyl or di-O-alkyl chains. The rate measurements indicate that the affinity of cholesterol for phospholipid does not vary significantly on change of the type of linkage (ether or ester) in phosphatidylcholine (PC) or of the positions of the fatty acyl chains in 1,2-diacyl-PC bearing one saturated and one unsaturated chain; furthermore, egg phosphatidylglycerol and egg phosphatidylethanolamine appear to have comparable affinities for cholesterol. However, the molecular packing in the bilayer and nearest-neighbor interactions involving cholesterol appear tightened more by N-palmitoylsphingomyelin than by dipalmitoyl-PC; on incorporation of 44 mol % of these phospholipids (which have the same fatty acyl chain composition) into either small or large unilamellar vesicles prepared with egg phosphatidylglycerol, the exchange rates were strikingly slower when the donor species contained sphingomyelin compared with PC. The rate of cholesterol exchange was 100% faster with small unilamellar vesicles than with large unilamellar vesicles as donors, suggesting that the looser packing in the highly curved small vesicles facilitates cholesterol desorption. The cholesterol exchange rate did not vary with the size of the acceptor vesicles, which indicates that desorption is the rate-limiting step in the exchange process in the presence of excess acceptors.     

Arsenate substitutes for phosphate in the human red cell sodium pump and anion exchanger

The sodium pump of human red blood cells mediates a Rb:Rb exchange that is dependent for maximal rates upon the simultaneous presence of intracellular ATP (or ADP) and phosphate. We have measured ouabain-sensitive 86Rb uptake into resealed ghosts of human red cells containing ADP and show that arsenate will substitute for phosphate in supporting the Rb:Rb exchange transport mode. The concentration dependence of arsenate-supported Rb:Rb exchange in ghosts containing 2 mM ADP shows both activating and inhibiting phases; the dependence upon phosphate shows similar characteristics. Elevation of the external [Rb] lowers the apparent affinity for arsenate since there is a shift to higher concentrations of arsenate in the activating and inhibiting phases of the arsenate concentration dependence curve. Similarly, elevation of [ADP] substantially reduces the inhibition of Rb:Rb exchange observed at higher [arsenate]. These effects are also observed in phosphate-supported Rb:Rb exchange. The phosphate requirement for Rb:Rb exchange involves phosphorylation of the sodium pump protein; the close agreement between the effects of arsenate and phosphate in supporting Rb:Rb exchange makes it likely that arsenylation of the sodium pump occurs during Rb:Rb exchange. Arsenate efflux from red blood cell ghosts into arsenate-free chloride medium is partially inhibited (77-80%) by DNDS (4,4'-dinitro-2,2'-stilbenedisulfonic acid), this compares with 82-87% inhibition by DNDS of phosphate efflux under the same conditions. It appears that Band III, the red cell anion transport system, accepts arsenate in a similar fashion to phosphate and that a fraction of the flux of both anions may occur through pathways other than Band III. Thus, in human red blood cells, both the sodium pump and the anion exchange transport system will accept arsenate as a phosphate congener and the protein-arsenate interactions are very similar to those with phosphate.     

Hypertriglyceridemia and hypercholesterolemia: effects of drug treatment on fatty acid composition of plasma lipids and membranes

The effect of atorvastatin, simvastatin and gemfibrozil on fatty acid composition of plasma phospholipids (PL), cholesterol esters (CE), triglycerides (TG) and red cell membrane ghosts (G) has been determined in appropriate sample populations of individuals with hypertriglyceridemia (HTG) or hypercholesterolemia (HCHL). Treatments were appropriate for the condition, gemfibrozil for HTG and a statin for HCHL. Modifications depend on the drug and lipid fraction examined. Both classes of drugs modify fatty acid composition but gemfibrozil modifications are more numerous and dramatic than are the modifications by statins. Gemfibrozil produces major modifications in fatty acid composition, which are both fatty acid and lipid class specific but generally decreases SFA and increases PUFA (mainly n6) and increases the proportion of fatty acids with chain length of 18C or more. Statins tend to increase chain length but have less effect on saturation. Notably, all three drugs increased arachidonic acid (AA) in PL and CE. Statins decreased gamma-linoleic acid (GLA) in PL and CE but gemfibrozil only increased GLA in TG.     

II. Phospholipid exchange and size enlargement in sonicated vesicles induced by a ‘critical’ fatty acid concentration

Size enlargement of dipalmitoyl phosphatidylcholine vesicles was greatly accelerated in the range of the phase-transition temperatures, when fatty acid concentration was above a threshold level (‘critical’ concentration). This ‘critical’ concentration varied with the length of the fatty acid chain. The size enlargement process had second-order kinetics dependent on the vesicle concentration. Alkaline pH and low ionic strength inhibited the rate of size enlargement.Phospholipid exchange between dimyristoyl and dipalmitoyl phosphatidylcholine vesicles increased abruptly above a ‘critical’ fatty acid concentration. The donor vesicles were those vesicles in which fatty acids reached the ‘critical’ concentration. The phospholipid exchange occurred both in fluid- and in solid-state vesicles. The ‘critical’ fatty acid concentration accelerating the phospholipid exchange process was lower than that accelerating the size enlargement process.The phospholipid exchange process explained in terms of a diminished hydrophobic attraction among the phospholipid molecules of the bilayer occurs via a free phospholipid molecule transfer through the aqueous phase. The size enlargement process is interpreted in terms of high fatty acid concentration in the membrane fluid domains. The membrane structure is locally perturbed inducing vesicle sticking after collision.     

Nature of the Elements Transporting Long-Chain Fatty Acids Through the Red Cell Membrane

Docosahexaenoic acid is found to be bound to three equivalent sites on albumin with the same affinities as palmitic acid at 0–38°C, which demonstrates that ethene-1,2-diyl- and methylene-groups contribute equally to the affinity. The equilibrium dissociation constants (K _dm s) for red cell membrane binding sites of linoleic- and docosahexaenoic acid at pH 7.3 are determined at temperatures between 0 and 37°C. The temperature-independent capacities for binding are 12 ± 1 and 25.4 ± 3.0 nmoles g⁻¹ ghosts respectively. Double isotope binding experiments reveal that the unsaturated fatty acids: arachidonic-, linoleic-, docosahexaenoic-, and oleic acid have partially shared capacities in ratios approximately 1:2:4:5, in contrast to the noncompetitive binding of palmitic acid. The observations suggest a two-tier binding limitation. One is the number of protein sites binding fatty acid anions electrostatically and the other is the number of suitable annular lipids adaptively selected among membrane lipids by the hydrocarbon chain. These competition conditions are confirmed by measurements of the tracer exchange efflux at near 0°C from albumin-free and albumin-filled ghosts of linoleic- and docosahexaenoic acid, either alone or in the presence of arachidonic- and palmitic acid. Under equilibrium conditions, the calculated ratios of inside to outside membrane binding is below 0.5 for four unsaturated fatty acids. The unidirectional rate constants of translocation between the inside and the outside correlate with the number of double bonds in these fatty acids, which are also correlated with the dissociation rate constants of the complexes with albumin. The membrane permeation occurs presumably by binding of the anionic unsaturated fatty acids to an integral protein followed by channeling of the neutral form between opposite binding sites of the protein through annular lipids encircling the protein. Received: 30 June 1997/Revised: 23 February 1998     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity

Background

Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure.

Methodology/Principal Findings

We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA^all-Ala). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles.

Conclusions/Significance

The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.     

Fatty acid-albumin complexes and the determination of the transport of long chain free fatty acids across membranes

Understanding the mechanism that governs the transport of long chain free fatty acids (FFA) across lipid bilayers is critical for understanding transport across cell membranes. Conflicting results have been reported for lipid vesicles; most investigators report that flip-flop occurs within the resolution time of the method (<5 ms) and that dissociation from the membrane is rate limiting, while other studies find that flip-flop is rate limiting and on the order of seconds. We have reinvestigated this problem and find that the methods used in studies reporting rapid flip-flop have not been interpreted correctly. We find that accurate information about transport of FFA across lipid vesicles requires that FFA be delivered to the vesicles as complexes with albumin (BSA). For example, we find that stopped-flow mixing of uncomplexed FFA with small unilamellar vesicles (SUV) containing pyranine yields the very fast influx rates reported previously (>100 s(-1)). However, these influx rates increase linearly with lipid vesicle concentration and can therefore not, as previously interpreted, represent flip-flop. In contrast, measurements of influx rates in SUV and giant unilamellar vesicles performed with oleate-BSA complexes reveal no dependence on vesicle concentration and yield influx rate constants of approximately 4 and approximately 0.5 s(-1), respectively. Rate constants for efflux and dissociation were determined from the transfer of oleate from vesicles to BSA and reveal similar influx and efflux but dissociation rate constants that are approximately 5-10-fold greater. We conclude that flip-flop is rate limiting for transport of FFA across lipid vesicles and slows with an increasing radius of curvature. These results, in contrast to those reporting that flip-flop is extremely fast, indicate that the lipid bilayer portion of biological membranes may present a significant barrier to transport of FFA across cell membranes.     

Quenching of red cell tryptophan fluorescence by mercurial compounds

Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.     

Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length

The kinetics and thermodynamics of the transmembrane movement (flip-flop) of fluorescent analogs of phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were investigated to determine the contributions of headgroup composition and acyl chain length to phospholipid flip-flop. The phospholipid derivatives containing n-octanoic, n-decanoic or n-dodecanoic acid in the sn-1 position and 9-(1-pyrenyl)nonanoic acid in the sn-2 position were incorporated at 3 mol% into sonicated single-bilayer vesicles of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC). The kinetics of diffusion of the pyrene-labeled phospholipids from the outer and inner monolayers of the host vesicles to a large pool of POPC acceptor vesicles were monitored by the time-dependent decrease of pyrene excimer fluorescence. The observed kinetics of transfer were biexponential, with a fast component due to the spontaneous transfer of pyrenyl phospholipids in the outer monolayer of labeled vesicles and a slower component due to diffusion of pyrenyl phospholipid from the inner monolayer of the same vesicles. Intervesicular transfer rates decreased approx. 8-fold for every two carbons added to the first acyl chain. Correspondingly, the free energy of activation for transfer increased approx. 1.3 kcal/mol. With the exception of PE, the intervesicular transfer rates for the different headgroups within a homologous series were nearly the same, with the PC derivative being the fastest. Transfer rates for the PE derivatives were 5-to 7-fold slower than the rates observed for PC. Phospholipid flip-flop, in contrast, was strongly dependent on headgroup composition with a smaller dependence on acyl chain length. At pH 7.4, flip-flop rates increased in the order PC less than PG less than PA less than PE, where the rates for PE were at least 10-times greater than those of the homologous PC derivative. Activation energies for flip-flop were large, and ranged from 38 kcal/mol for the longest acyl chain derivative of PC to 25 kcal/mol for the PE derivatives. Titration of the PA headgroup at pH 4.0 produced an approx. 500-fold increase in the flip-flop rate of PA, while the activation energy decreased 10 kcal/mol. Increasing acyl chain length reduced phospholipid flip-flop rates, with the greatest change observed for the PC analogs, which exhibited an approx. 2-fold decrease in flip-flop rate for every two methylene carbons added to the acyl chain at the sn-1 position.(ABSTRACT TRUNCATED AT 400 WORDS)     

Scavenger receptor-mediated uptake and metabolism of lipid vesicles containing acidic phospholipids by mouse peritoneal macrophages

We studied the mechanism of uptake and metabolism of exogenous phospholipids in mouse peritoneal macrophages using vesicles composed of various phospholipids and cholesterol. Macrophages in culture were found to actively incorporate and metabolize phosphatidylcholine/cholesterol vesicles containing small amounts of acidic phospholipids such as phosphatidylserine, phosphatidylinositol, or phosphatidic acid and to store the fatty acyl chains and cholesterol in triacylglycerol and cholesteryl ester form in their cytosol. These cells exhibited massive amounts of oil red O-positive lipid droplets, a typical feature of foam cells. The metabolism of exogenous phospholipid vesicles was completely inhibited by chloroquine and cytochalasin B, suggesting that vesicle uptake occurs by endocytosis. A similar type of metabolism was observed in guinea pig peritoneal macrophages, macrophage cell line J774.1, but not in Swiss 3T3 fibroblasts. Competition studies using various ligands for the scavenger receptor showed that acetylated low density lipoprotein (acetyl-LDL), dextran sulfate, or fucoidan was able to compete for up to 60% of the binding of phosphatidylserine-containing vesicles, and that copper-oxidized LDL (oxidized LDL) competed for more than 90% of the vesicle binding. On the other hand, phosphatidylserine-containing vesicles was able to compete for more than 90% of the binding of acetyl-LDL. These results indicate that acidic phospholipids are recognized by the scavenger receptors on the surface of macrophages and that more than one scavenger receptor exists on mouse peritoneal macrophages, i.e. one capable of recognizing acetyl-LDL, oxidized LDL, and an array of acidic phospholipids on membranes, and the other recognizing both acidic phospholipids and oxidized LDL but not acetyl-LDL.     

ATP-induced endocytosis in human erythrocyte ghosts. Characterization of the process and isolation of the endocytosed vesicles.

ATP-induced endocytosis in human erythrocyte ghosts has been studied, and a procedure for the isolation of the endocytotic vesicles is described. Under isotonic conditions and 37 degrees C, optimal endocytosis occurs with concentrations of 4 to 10 mM MgATP. Within 30 min, up to 45% of the membrane is removed from the surface and converted into sealed inside-out vesicles. Local anesthetics, such as chlorpromazine, potentiate ATP-induced endocytosis in ghosts. Forcing cells containing endocytotic vesicles through a hypodermic needle leads to the exclusive fragmentation of the outermost plasma membrane. The endocytosed vesicles can then be separated from these fragments by centrifugation on a gradient of dextran T70. Biochemical analyses indicate that endocytotic vesicles contain full complements of the major membrane proteins (i.e. also spectrin and actin), common phospholipids, fatty acids, and cholesterol. Furthermore, they exhibit a fully intact spectrin component 2 phosphorylation machinery. In contrast, MgATPase activity is largely excluded from these vesicles. The novel inside-out vesicles described have properties different from those of previously analyzed fragments of the erythrocyte membrane. They will permit a detailed study of a native spectrin-actin network now exposed to the outside.     

An activation-collision mechanism for cholesterol transfer between membranes

We report the results of experiments which show that cholesterol transfer between membranes cannot proceed by aqueous diffusion, as widely held, but must involve a more complex mechanism. (a) The rate of transfer of [3H]cholesterol from red blood cells was found to vary inversely with the size of the acceptor particle (ghosts, vesicles of ghosts, liposomes, and plasma lipoproteins). (b) The transfer of [3H]cholesterol from red blood cells to ghosts was accelerated by the presence of plasma, even though the plasma competed with the ghosts as an acceptor. (c) The rate of transfer of [3H]cholesterol from red blood cells to ghosts decreased to zero with increasing dilution but was not simply second-order. (d) The cholesterol in retinal rod disc membranes is not at equilibrium with plasma lipoproteins in that disc cholesterol increased when the homogenates were incubated in vitro with plasma. (e) The kinetics of cholesterol transfer cannot be limited by unstirred layer effects since the transfer of lysolecithin in the same system was faster than that of cholesterol by 3 orders of magnitude. The simplest model compatible with all the data suggests a two-step pathway involving a first-order followed by a second-order process. The first step could be a unimolecular activation event, perhaps the movement of the sterol in the donor particle to a more exposed (hydrated) position. In the second step, the activated sterol would be transferred during transient collisions between donor and acceptor particles. When collision is not rate-limiting, the overall process would appear to be simply first-order, hence kinetically indistinguishable from the aqueous diffusion mechanism. The activation-collision model thus not only rationalizes our data but is also consistent with the simpler kinetics previously reported for the transfer of both membrane phospholipids and sterols.     

Intrinsic segments of band 3 that are associated with anion transport across red blood cell membranes

Summary After treatment of red cell ghosts with chymotrypsin, the predominant intrinsic peptides remaining in the membrane fraction are 15,000 and 9,000 daltons mol wt. After partial extraction with Triton X-100, the residual membrane vesicles have almost no other stained peptides and such vesicles are reported to carry out anion transport activities sensitive to specific inhibitors. In vesicles derived from cells treated with DIDS(4,4-diisothiocyano-2,2-stilbene disulfonic acid), an irreversible inhibitor of anion transport that is highly localized in an abundant intrinsic protein known as band 3, the probe is largely recovered in the 15,000 dalton peptide. The part of band 3 from which it is derived is a previously reported 17,000 transmembrane segment (Steck, T.L., Ramos, R., Strapazon, E., 1976,Biochemistry 15:1154). The 9,000-dalton peptide is present in the vesicles in a one-to-one mole ratio with the 15,000-dalton peptide, suggesting that both are derived from the same protein. This conclusion is supported by the finding that the 35,000-dalton C-terminal end of band 3, derived by chymotrypsin treatment of cells, is further proteolysed if the cells are converted to ghosts and its disappearance coincides with the appearance of the 9,000-dalton fragment. Evidence is presented that the 9,000-dalton fragment crosses the bilayer and that it is closely associated with the 15,000-dalton peptide.This paper is dedicated to the memory of Walther Wilbrandt.