Immobilization of phospholipid vesicles on alkyl derivatives of agarose gel beads

We have immobilized phospholipid vesicles on hydrophobic derivatives of agarose gel beads. The vesicles were prepared from cholate-solubilized egg yolk phospholipids by gel filtration in 0.2 M NaCl at pH 7.1, which produced small vesicles, or in 0.5 M (NH₄)₂SO₄ at pH 8.0, which yielded large ones. The small vesicles eluted with K_d 0.4–0.6 and the large ones with K_d 0.05 on Sepharose 4B. Butyl, octyl and dodecyl sulfide derivatives of Sepharose 4B were synthesized using 1,4-butanediol diglycidyl ether and alkyl mercaptans (Maisano, F., Belew, M. and Porath, J. (1985) J. Chromatogr. 321, 305–317). The phospholipid vesicles were immobolized on 0.6–1-ml columns of these adsorbents in the salt solution that had been used for the preparation of the liposomes. A ligand concentration of 8 μmol per ml gel was sufficient for immobilization of small as well as large vesicles. The capacity of immobilization per ml gel was at least 20–100 and 1.5–3 μmol of phospholipids for small and large vesicles, respectively. The rate of adsorption of small vesicles was initially 0.3–0.5 μmol of phospholipids per min per ml gel, but decreased later to 0.2–0.3 μmol/min per ml as the gel bead surfaces approached saturation. These rates were determined at a vesicle concentration corresponding to 1.2 mM phospholipids and at room temperature. The butyl adsorbent gave a higher initial adsorption rate but a lower capacity than the dodecyl adsorbent, probably due to differences in the energy thresholds for ligand penetration through the hydrophilic surface layer of the vesicles, and to differences in the binding strength. The maximal concentration of adsorbed small vesicles that we achieved, 100 μ mol of phospholipids per milliliter octyl surfide-Sepharose 4B, would be equivalent to close-packing of the spherical phospholipid vesicles in 40% of the accessible volume of the gel beads.     

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Interactions among red cell membrane proteins

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.     

Depletion of membrane skeleton in red blood cell vesicles.

A possible physical interpretation of the partial detachment of the membrane skeleton in the budding region of the cell membrane and consequent depletion of the membrane skeleton in red blood cell vesicles is given. The red blood cell membrane is considered to consist of the bilayer part and the membrane skeleton. The skeleton is, under normal conditions, bound to the bilayer over its whole area. It is shown that, when in such conditions it is in the expanded state, some cell shape changes can induce its partial detachment. The partial detachment of the skeleton from the bilayer is energetically favorable if the consequent decrease of the skeleton expansion energy is larger than the corresponding increase of the bilayer-skeleton binding energy. The effect of shape on the skeleton detachment is analyzed theoretically for a series of the pear class shapes, having decreasing neck diameter and ending with a parent-daughter pair of spheres. The partial detachment of the skeleton is promoted by narrowing of the cell neck, by increasing the lateral tension in the skeleton and its area expansivity modulus, and by diminishing the attraction forces between the skeleton and the bilayer. If the radius of the daughter vesicle is sufficiently small relative to the radius of the parent cell, the daughter vesicle can exist either completely underlaid with the skeleton or completely depleted of the skeleton.     

Transbilayer distributions of red cell membrane phospholipids in unilamellar vesicles

The phospholipid organization in unilamellar vesicles comprised of various purified phospholipid components of monkey erythrocyte membrane was ascertained using phospholipase A2 and trinitrobenzenesulfonic acid as external membrane probes. The vesicles were formed by sonication or detergent dialysis and fractionated by centrifugation or gel permeation chromatography. Experiments were done to confirm that the phospholipase A2 treatments did not cause lysis or induce fusion of the vesicles. This enzyme hydrolysed only the glycerophospholipids in the outer surface of the vesicles. The amounts of the external phospholipids determined by this enzymatic method were verified using the chemical probe, trinitrobenzenesulfonic acid. The choline-containing phospholipids and phosphatidylethanolamine localized randomly in the two surfaces of sonicated vesicles (outer diameter, about 30 nm), whereas phosphatidylserine preferentially distributed in the inner monolayer. This phosphatidylserine asymmetry virtually disappeared in detergent dialysed vesicles (outer diameter, about 45 nm). Furthermore, inclusion of cholesterol in both the types of vesicles resulted in more random glycerophospholipid distributions across the plane of vesicles bilayer, presumably due to the cholesterol-induced increases in the size of vesicles. These results demonstrate that the transbilayer distribution of erythrocyte membrane phospholipids in unilamellar vesicles are controlled mainly by the surface curvature rather than by interlipid interactions, and therefore suggest that phospholipid-phospholipid and phospholipid-cholesterol interactions should not play any significant role in determining the membrane phospholipid asymmetry in red cells. It is proposed that this asymmetry primarily originates from differential bindings of phospholipids with membrane proteins in the two leaflets of the membrane bilayer.     

Interaction of microtubule proteins with phospholipid vesicles

We have examined the interaction of unilamellar dimyristoyl phosphatidylcholine liposomes with the high-speed supernate of brain homogenate and with tubulin purified through one or two cycles of microtubule assembly-disassembly. Tubulin and certian high molecular weight proteins are selectively adsorbed from these mixtures onto liposomes. The composition of adsorbed proteins is similar to that obtained during corresponding cycles of microtubule assembly, suggesting the equivalency of these processes. Adsorption induces stacking and/or fusion of liposomes into multilamellar structures indicating strong protein-lipid interaction. In addition, liposome-adsorbed tubulin forms extensive intermolecular disulfide bridges that are inert to reducing agents in the aqueous medium. The observations form a basis for further study of the distribution, function, and properties of membrane-bound tubulin.     

Change in membrane fluidity induced by lectin-mediated phase separation of the membrane and agglutination of phospholipid vesicles containing glycopeptides

Changes in membrane fluidity induced by lectin addition to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles containing synthetic glycopeptides were measured by depolarization of the fluorescent probes 8-anilino-1-naphthalenesulfonate (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). In the present synthesized glycopeptides, N-acetylglucosamine (GlcNAc) and a tripeptide were connected by aliphatic chains of different lengths. A pyrenyl group, which is introduced to the peptide moiety, acted as a probe to characterize the distribution of glycopeptides in the membrane on the basis of its excimer formation. The glycopeptide was shown to be distributed to DPPC vesicles with the peptide moiety buried in the hydrophobic core of the lipid bilayer and the glyco moiety exposed to the outside of the membrane. By the addition of wheat germ agglutinin (WGA) to the vesicles containing the glycopeptides, intravesicular cross-linking of glycopeptides in the membrane and aggregation of vesicles were observed. The intravesicular cross-linking was antagonized by GlcNAc above the phase transition temperature. However, the dissociation of aggregation required the addition of a stronger antagonist, N,N'-diacetylchitobiose. The addition of the glycopeptide to DPPC vesicles above the phase transition temperature decreased the membrane fluidity. However, a succeeding addition of WGA caused a large increase of membrane fluidity at either the surface or the hydrophobic core of the lipid bilayer membrane. This increase of membrane fluidity was attributed to two factors by use of two kinds of antagonists having different potencies: one is a WGA-mediated cross-linking of glycopeptides in the membrane, and the other is a close contact of vesicles on aggregation.     

Water-soluble proteins of the human red cell membrane

Summary Procedures were developed for preparation of red cell membranes almost free of hemoglobin but with minimal loss of membrane proteins. Two water-soluble protein fractions are described, each constituting about 25% of the ghost protein. The first is ionically bonded and can be solubilized in water rapidly at pH 7.0 and more slowly at higher ionic strength solutions, with a minimal rate at 20mm. This fraction contains four major components with molecular weights ranging from 30,000 to 48,000. The second fraction can only be solubilized at an appreciable rate if Ca⁺⁺ is absent and at higher pH (9.0). It is predominantly a single molecular weight component (150,000). It tends to aggregate at higher ionic strength and in the presence of Ca⁺⁺. Evidence is presented suggesting that the water-soluble proteins are present at the inner face of the membrane. The lipids remain in a water-insoluble residue that contains four major protein components ranging in molecular weight from 30,000 to 100,000. The latter is the predominant component. Only the residue contains the Na⁺–K⁺-activated ATPase, the cholinesterase, antigenic activity and most of the sialic acid and carbohydrate. The first water-soluble fraction contains a Mg⁺⁺-activated ATPase. The extraction of the water-soluble proteins is accompanied by anatomical changes resulting finally in the formation of small membranous vesicles.     

Chemically-induced cation permeability in red cell membrane vesicles. The sidedness of the response and the proteins involved.

Cation fluxes were measured in right-side-out and inside-out vesicles obtained from human red cells. Rubidium, which is spontaneously released at very slow rates, can be rapidly released from both types of vesicle by addition of valinomycin. P-Chloromercuriphenyl sulfonic acid (PCMBS) also increases the cation permeability of the vesicles with reversal to normal after addition of dithiothreitol. The effect of PCMBS is considerably larger and appears faster in the inside-out vesicles as compared to the right-side-out vesicles, the difference being greater at low temperatures. These data indicate that the SH groups responsible for the changes in cation permeability are more accessible from the inside face of the membrane. The response to PCMBS was not diminished after selective removal of extrinsic proteins by alkaline extraction, and/or after the membranes were exposed to proteolytic enzymes. The major polypeptide component remaining in vesicles after both treatments was a 17 000-dalton transmembrane fragment derived from band 3 which might, therefore, be responsible for the permeability response. Addition of Ca2+ to either right-side-out or inside-out vesicles, in the presence or absence of ionophore A23187, was without effect on monovalent cation permeability, indicating that the mechanism of Ca2+-induced K+ permeation was lost or inactivated during the preparation of the vesicles.     

Interaction of divalent cations and proteins with phospholipid vesicles

The broadening of spin-label absorption lines resulting from spin-exchange reactions that occur during collision with paramagnetic Ni2+ is diminished when Ni2+ binds to phospholipid vesicles. Subsequent addition of non-paramagnetic ions that compete for binding sites releases Ni2+ into solution and restores the line-broadening. The concentrations of various ions required to achieve this effect was used to order the ions with respect to their binding to vesicles containing phosphatidylethanolamine and phosphatidylglycerol. The relative strengths of binding for those ions studied were: Ca2+ > Mg2+ > Zn2+ > Sr2+ > Ba2+. The spin-broadening assay was also used to study the effects of two proteins on the availability of Ni2+-binding sites on the vesicles. Ribonuclease, which is thought to associate electrostatically as an extrinsic protein on the surface of vesicles, completely blocked the Ni2+-binding sites at comparatively low protein concentrations. Quantitative considerations of these data suggest the possibility that Ni2+ may bind preferenetially to phosphatidylglycerol, and that these binding sites are aggregated in the ribonuclease-containing vesicles. In contract to ribonuclease, cytochrome c does not block Ni2+-bindings sites on the phospholipid vesicles, but rather contains sites of its own that bind Ni2+, both when the protein is in solution and when it is associated with the vesicles. These results are consistent with other studies which suggest that cytochrome c becomes partially embedded in membrane bilayers and associates with phospholipid molecules through hydrophobic interactions.     

Insulin-dependent phosphorylation of GTP-binding proteins in phospholipid vesicles

The involvement of GTP-binding proteins (G proteins) in insulin action has been investigated in an in vitro system. Insulin receptors that have been purified by wheat germ lectin chromatography and either tyrosine-agarose chromatography, sucrose density centrifugation, or insulin-Sepharose chromatography have been co-inserted into phospholipid vesicles with different purified G proteins. The results of these studies indicate that a specific insulin-promoted phosphorylation of two G proteins, Go and Gi, can occur in these phospholipid vesicles. Bovine retinal transducin is a poor substitute for Go and Gi, being only weakly phosphorylated by the insulin receptor, and bovine brain Gs is not a substrate. The phosphorylation of Gi and Go occurs primarily on the alpha-subunits. Under optimal conditions, about one alpha o- or alpha i-subunit is phosphorylated on a tyrosine residue for every two beta-subunits of the insulin receptor, suggesting a 1:1 interaction between these G proteins and the heterotetrameric (alpha 2 beta 2) insulin receptor molecular. The inactive (GDP-bound) form of the alpha-subunits appears to be the preferred substrate, with the phosphorylation being significantly reduced in alpha o and alpha i upon the binding of guanosine 5'-O-thiotriphosphate (GTP gamma S) and completely eliminated in the pure alpha-GTP gamma S complex of transducin. The Gi and Go proteins also cause an enhancement of the insulin-stimulated receptor autophosphorylation. This enhancement is a reflection of an increased incorporation of the insulin receptor into lipid vesicles which is induced by these G proteins. Taken together these results provide evidence for the interactions of G proteins with the insulin receptor in a lipid milieu.     

Effects of superoxide anions on red cell deformability and membrane proteins.

The effect of superoxide anions (O2-) on red blood cells (RBC) deformability and membrane proteins was investigated using hypoxanthine-xanthine oxidase system. Exposure of RBC to O2- caused a marked decrease in RBC deformability with a concomitant increase in cell volume and shape changes. The RBC exposed to O2- also displayed pronounced degradation of membrane proteins such as band 3 protein and spectrin; new bands of low molecular weight products appeared as the original membrane proteins tended to diminish, without the appearance of high molecular weight products. Since the membrane proteins are involved in processes regulating membrane properties such as permeability and viscoelasticity, the decreased deformability induced by O2- may be attributable to changes in membrane proteins. Interestingly, resealed ghosts exposed to O2- did not show any significant change in membrane proteins, which suggests the existence of further generation of O2- and subsequent production of other active oxygen species mediated by O2(-)-initiated autoxidation of hemoglobin in intact RBC. Furthermore, electrophoretic analysis suggested that active oxygens increased the endogenous proteolytic susceptibility of RBC. In conclusion, a close linkage was suggested between RBC deformability and the membrane proteins.     

Lectins facilitate calcium-induced fusion of phospholipid vesicles containing glycosphingolipids

Ca2+-induced fusion of phospholipid vesicles containing globoside (GL-4) or disialoganglioside (GDla) is several-fold slower than the fusion of the pure phospholipid vesicles. Lectins specific for these glycosphingolipids, soybean agglutinin and wheat germ agglutinin, respectively, enhance the rate of fusion when added to the vesicle suspension before the introduction of Ca2+. The enhancement depends on the lectin concentration and the time of preincubation with the lectin. We propose that lectins facilitate membrane fusion by inducing intermembrane contact, which is the first step in the overall process of membrane fusion, or by laterally phase separating the inhibitory glycolipids.     

Characteristics and regulation of active calcium transport in inside-out red cell membrane vesicles

Sealed, inside-out human red cell membrane vesicles, prepared by a modified method of Steck (Steck T.L. (1974) in Methods in Membrane Biology (Korn, E.D., ed.), Vol 2, pp. 245–281, Plenum Press, New York), accomplish an ATP and Mg²⁺-dependent uphill calcium uptake with a reproducible maximum rate of 12–15 nmol/mg vesicle protein per min under physiological conditions. This maximum rate is increased by about 60–70% in the presence of a heatstable cytoplasmic activator protein (calmodulin) obtained from red cells. Calcium efflux from inside-out vesicles is smaller than 0.01 nmol/mg vesicle protein per min at intravesicular calcium concentrations between 0.1 and 20.0 mM.In the presence of Mg²⁺, active calcium uptake is supported by ATP, ITP, or UTP, but not by ADP, AMP, or p-nitrophenyl phosphate. The optimum pH for the process is 7.4–7.6, and the activation energy is 19–20 kcal/mol, irrespective of the presence or absence of calmodulin. Calcium uptake in inside-out vesicles is unaffected by ouabain or oligomycin, but blocked by low concentrations of lanthanum, ruthenium red, quercetin and phloretin. K⁺ and Na⁺, when compared to choline⁺ or Li⁺, significantly increase active calcium uptake. This stimulation by K⁺ and Na⁺ is independent of that by calmodulin.Concentrated red cell cytoplasm activates calcium uptake at low soluble protein:membrane protein ratios, while a ‘deactivation’ of the transport occurs at high cytoplasm: membrane protein ratios. A heat-labile cytoplasmic protein fraction antagonizing calmodulin activation, can be separated by DEAE-Sephadex chromatography. Based on these findings the regulation of active calcium transport in human red cells is discussed.     

Use of octyl beta-thioglucopyranoside in two-dimensional crystallization of membrane proteins

A great interest exists in producing and/or improving two-dimensional (2D) crystals of membrane proteins amenable to structural analysis by electron crystallography. Here we report on the use of the detergent n-octyl beta-d-thioglucopyranoside in 2D crystallization trials of membrane proteins with radically different structures including FhuA from the outer membrane of Escherichia coli, light-harvesting complex II from Rubrivivax gelatinosus, and Photosystem I from cyanobacterium Synechococcus sp. We have analyzed by electron microscopy the structures reconstituted after detergent removal from lipid-detergent or lipid-protein-detergent micellar solutions containing either only n-octyl beta-d-thioglucopyranoside or n-octyl beta-d-thioglucopyranoside in combination with other detergents commonly used in membrane protein biochemistry. This allowed the definition of experimental conditions in which the use of n-octyl beta-d-thioglucopyranoside could induce a considerable increase in the size of reconstituted membrane structures, up to several micrometers. An other important feature was that, in addition to reconstitution of membrane proteins into large bilayered structures, this thioglycosylated detergent also was revealed to be efficient in crystallization trials, allowing the proteins to be analyzed in large coherent two-dimensional arrays. Thus, inclusion of n-octyl beta-d-thioglucopyranoside in 2D crystallization trials appears to be a promising method for the production of large and coherent 2D crystals that will be valuable for structural analysis by electron crystallography and atomic force microscopy.     

Adhesion of phospholipid vesicles to Chinese hamster fibroblasts: Role of cell surface proteins

The adhesion of artificially generated lipid membrane vesicles to Chinese hamster V79 fibroblasts in suspension was used as a model system for studying membrane interactions. Below their gel-liquid crystalline phase transition temperature, vesicles comprised of dipalmitoyl lecithin (DPL) or dimyristoyl lecithin (DML) absorbed to the surfaces of EDTA- dissociated cells. These adherent vesicles could not be removed by repeated washings of the treated cells but could be released into the medium by treatment with trypsin. EM autoradiographic studies of cells treated with[(3)H]DML or [(3)H]DPL vesicles showed that most of the radioactive lipids were confined to the cell periphery. Scanning electron microscopy and fluorescence microscopy further confirmed the presence of adherent vesicles at the cell surface. Adhesion of DML or DPL vesicles to EDTA-dissociated cells modified the lactoperoxidase-catalyzed iodination pattern of the cell surface proteins; the inhibition of labeling of two proteins with an approximately 60,000- dalton mol wt was particularly evident. Incubation of cells wit h (3)H-lipid vesicles followed by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis showed that some of the (3)H-lipid migrated preferentially with these approximately 60,000-mol wt proteins. Studies of the temperature dependence of vesicle uptake and subsequent release by trypsin showed that DML or DPL vesicle adhesion to EDTA- dissociated cells increased with decreasing temperatures. In contrast, cells trypsinized before incubation with vesicles showed practically no temperature dependence of vesicle uptake. These results suggest two pathways for adhesion of lipid vesicles to the cell surface-a temperature-sensitive one involving cell surface proteins, and a temperature-independent one. These findings are discussed in terms of current models for cell-cell interactions.