Morphology of haemoglobin-containing human erythrocyte ‘ghosts’

Resealed erythrocyte ‘ghosts’ have been proposed as in vivo carriers for enzymes in the therapy of inherited metabolic diseases. A long life-span of this carrier is required when the erythrocyte ‘ghost’ is intended to be the site of substrate degradation in the circulation. Erythrocyte ‘ghosts’ have been prepared that show cell content and membrane transport characteristics that are closely similar to those of normal erythrocytes. Since the morphology of these ‘ghosts’ could probably also affect the in vivo life-span, haemoglobin-containing human erythrocyte ‘ghosts’ have been studied using scanning electron-microscopy.Preparation of the erythrocyte ‘ghosts’ involved a method of hypo-osmotic dialysis and consecutive iso-osmotic dialysis. Samples for scanning electron microscopy were prepared by fixation in glutaraldehyde, with post-fixation in osmium tetroxide. Dehydration was obtained by increasing concentrations of ethanol and critical-point-drying. Control experiments with normal erythrocytes showed no major artefacts by the methods used. Erythrocyte ‘ghosts’ showed polymorphic shapes. Two thirds were stomatocytes, but echinocytes and cells with deep invaginations or inter-twisted infoldings were also observed. Distribution of the different cell types could be affected by the preparation techniques used. Washing of the erythrocyte ‘ghosts’ at very low centrifugation speeds resulted in 60% of the cells appearing as biconcave discoids and one third as stomatocytes.The results demonstrate that the preparation disturbed the balance of the biconcave shape of resealed erythrocyte ‘ghosts’. Minor alterations of the methods allowed the preparation of erythrocyte ‘ghosts’, the majority of which showed morphology closely similar to that of normal erythrocytes. Attention must be given to all preparation factors to avoid irreversible damage and therefore prior to use of these cells in patients or when methods are altered, the morphology of these cellular carriers must be closely controlled.     

A quantitative study of electroporation showing a plateau in net molecular transport.

Electroporation is believed to involve a temporary structural rearrangement of lipid bilayer membranes, which results in ion and molecular transport across the membrane. The results of a quantitative study of molecular transport due to electroporation caused by a single exponential pulse are presented; transport of four molecules of different physical characteristics across erythrocyte ghost membranes is examined as a function of applied field strength. Flow cytometry is used to quantitatively measure the number of molecules transported for 10(4) to 10(5) individual ghosts for each condition. This study has four major findings: 1) Net transport first increases with field strength, but reaches a plateau at higher field strengths. Significant transport is found at or below 1 kV/cm, and transport plateaus begin at field strengths between 2 and 5 kV/cm depending on the molecule transported. 2) A single population of ghosts generally exists, but exhibits a wide distribution in the amount of molecular transport. 3) Under the conditions used, the direction of transport across the ghost membrane does not appear to affect molecular transport significantly. 4) Large numbers of ghosts may be destroyed by the electroporation procedure.     

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes

The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N-ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p-(chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport.     

Absence of transbilayer diffusion of spin-labeled sphingomyelin on human erythrocytes. Comparison with the diffusion of several spin-labeled glycerophospholipids

We have measured the transbilayer diffusion at 4 degrees C of spin labeled analogs of sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid in the human erythrocyte membrane. Measurements were also carried out in ghosts, released without ATP, and on large unilamellar vesicles made with total lipid extract. As reported previously (Seigneuret, M. and Devaux, P.F. (1984) Proc. Natl. Acad. Sci. USA 81, 3751-3755), the amino phospholipids are rapidly transported from the outer to the inner leaflet on fresh erythrocytes, whereas phosphatidylcholine diffuses slowly. We now show that phosphatidic acid behaves like phosphatidylcholine: approximately 10% is internalized in 5 h at 4 degrees C. Under the same experimental conditions, no inward transport of sphingomyelin can be detected. In ghosts resealed without ATP, all glycerophospholipids tested diffuse slowly from the outer to the inner leaflet (approx. 10% in 5 h) while no transport of sphingomyelin is seen. Finally in lipid vesicles, the inward diffusion of all glycerophospholipids is less than 2% in 5 h and a very small transport of sphingomyelin can be measured. These results confirm the existence of a selective inward aminophospholipid transport of fresh erythrocytes and suggest a slow and passive diffusion of all phospholipids on ghosts, resealed without ATP, as well as on lipid vesicles.     

Membrane transport of anandamide through resealed human red blood cell membranes

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments at 0 degrees C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 +/- 0.023, and the rate constant of unidirectional flux from inside to outside is 0.361 +/- 0.023 s(-1). The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]o) increases with the square root of [BSA]o in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane very rapidly, within seconds. At a molar ratio of anandamide to BSA of <1, membrane binding of anandamide increases with increasing temperatures between 0 degrees C and 37 degrees C, and the equilibrium dissociation constants are in the nanomolar range. The nature of membrane binding and the mechanism of membrane translocation are discussed.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

Participation of a transmembrane proton gradient in 5-hydroxytryptamine transport by platelet dense granules and dense-granule ghosts.

Dense granules, the storage organelles for 5-hydroxytryptamine in blood platelets, have been isolated from porcine platelets and are shown to transport 5-hydroxytryptamine in response to a transmembrane proton gradient (delta pH). Transport in the absence of delta pH is minimal, and it is shown that a rapid increase in transport takes place as delta pH increases. Direct measurements with [14C]methylamine show a delta pH of 1.1 units (acid inside) for intact granules. Osmotically active ghosts of dense granules from which 95% of the endogenous 5-hydroxytryptamine content has been released have also been prepared. Ghosts swell in the presence of ATP and Mg2+, and this swelling is shown to be due to the entry of protons via a process linked to ATP hydrolysis. Proton entry is also apparently linked to anion penetration in ghosts. Steady-state 5-hydroxytryptamine transport in ghosts is stimulated approx. 3-fold on the addition of ATP to the incubation medium, and the stimulation of 5-hydroxytryptamine transport in ghosts correlates with the formation of a transmembrane delta pH. Ghosts generate a delta pH of 1.1-1.3 pH units (acid inside) in the presence of 5 mM-ATP/2.5 mM-MgSO4. delta pH is generated within 3 min at 37 degrees C and is dissipated by the ionophore nigericin and by NH4Cl. It is shown that an Mg2+-stimulated ATPase activity is present on the ghost membrane, and inhibition of the ATPase leads to a corresponding decrease in 5-hydroxytryptamine transport. The results presented support the idea that 5-hydroxytryptamine transport into platelet dense granules is dependent on the presence of a transmembrane delta pH and, together with previous findings by others, suggest a generalized mechanism for biogenic amine transport into subcellular storage organelles.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

Ca2+ and proton transport in chromaffin granule membranes: a proton NMR study

High-resolution proton NMR spectroscopy has been used to monitor the internal pH of chromaffin granule ghosts during Ca2+ influx through the membrane. For this purpose, ghosts were prepared by lysing and resealing chromaffin granules in a medium containing the disodium-ethylenediaminetetraacetic acid complex (Na2.EDTA). Uncomplexed EDTA and Ca.EDTA give rise to distinct sets of methylene peaks in the proton NMR spectrum. Free EDTA titrates with a pK near 6.6 in deuterated media; the chemical shifts that accompany titration have been used to monitor intravesicular pH changes which occur inside chromaffin granule ghosts as a result of ATPase activity and deprotonation of EDTA during Ca2+ influx and complex formation. ATPase activity results in an NMR-detectable proton gradient which is dissipated by nigericin. Experiments monitoring Ca2+ uptake showed that protons which are liberated inside ghosts as a result of Ca.EDTA complex formation are not extruded from the ghosts via a process coupled to Ca2+ entry. This suggests that the Ca2+ transport system of the chromaffin granule membrane occurs without concurrent proton antiport and is not directly coupled energetically to the transmembrane pH gradient.     

Sugar transport in reversibly hemolyzed avian erythrocytes

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37°C, 88% of the ghosts regained their permeability barrier to l-glucose. In these ghosts, the carrier-mediated rate of entry of $\text{3-O-}\text{methylglucose}$ was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Preparative isolation of Band 3, the predominant polypeptides of the human erythrocyte membrane

Band 3 proteins are the predominant polypeptide components of the human erythrocyte membrane and have been implicated in various transport activities. Following extraction of membrane ghosts with dimethylmaleic anhydride to remove two polypeptides (Bands 4.2 and 6) associated with Band 3, Band 3 proteins were solubilized along with the other major membrane glycoproteins (PAS 1–3) with Triton X-100 under nondenaturing conditions. Band 3 proteins were then purified (>95%) on a large scale by chromatography via thioldisulfide interchange on activated thiol-Sepharose 4B [agarose-(glutathione-2-pyridyl disulfide) conjugate]. This procedure allows the preparation of 20 to 25 mg of purified Band 3 proteins in high yield (>80%) from ghosts in a soluble form suitable for physical, chemical, and functional characterization.     

Sealed reticulocyte ghosts. An experimental model for the study of Fe2+ transport.

Sealed right-side-out reticulocyte ghosts transported and accumulated iron offered as 59Fe(2+)-ascorbate (Km = 1.1 microM). The uptake of iron by ghosts presented the characteristics of a transporter-mediated process: it responded to osmotic challenge, the rate of transport increased when iron was present in the opposing side, and the transport rate showed the temperature dependence typical of membrane-mediated processes. The transport of iron was dependent on an associated influx of Cl- in order to keep electroneutrality. Other transition metals, such as Cu2+, Zn2+, and Co2+, inhibited the transport of Fe2+. The overall characteristics of the system make reticulocyte sealed ghosts a very useful model in determining the basic mechanisms of membrane iron transport.     

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.     

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.     

Characterization of ion channels involved in the penetration of phage T4 DNA into Escherichia coli cells

The hypothesis of a channel-mediated transport of phage DNA into Escherichia coli cytoplasmic membrane has been formulated for a long time. In this paper, we present experimental evidence in favor of this proposal. We have analyzed the kinetics of the K+ efflux induced by T4 phage and ghosts (phage depleted of DNA) using a potassium selective electrode. We show that the K+ efflux is not catalyzed by the K+ transport systems. The Km of K+ efflux is the same for phage and ghosts. The rate of K+ efflux is linearly related to the multiplicity of infection. This suggests that phage and ghosts induce the formation of similar channels and that one channel is induced by one virion. The K+ efflux is associated with an influx of H+ and Na+ or Li+ which compete for entry through the channel. These ion fluxes may be responsible for the cell depolarization. The phage-induced channels allow the passage of DNA. They are only transiently opened, and their closing leads to cellular repolarization. The ghost-induced channels remain open. The insertion and conformation of the channels in the membrane depend on the temperature and their confirmation is voltage-dependent. We give an estimate of their size.     

Palmitate binding to and efflux kinetics from human erythrocyte ghost

At 0 degrees C, pH 7.3, palmitate (PA) binds to human erythrocyte ghosts suspended in 0.2% bovine serum albumin (BSA) solution with molar ratios of PA to BSA, v, between 0.2 and 1.3. The binding depends on the water phase PA concentration, measured in equilibrium experiments, using BSA-filled ghosts as semipermeable bags. The saturable binding has a capacity of 19.4 +/- 7.5 nmol g-1 packed ghosts (7.2 x 10(9) cells) and Kd = 13.5 +/- 5 nM. PA exchange efflux kinetics to 0.2% BSA is recorded from ghosts without and with 0.2% BSA with a resolution time of about 1 s. Data are analyzed in terms of compartmental models. Using BSA-free ghosts the kinetics is essentially monoexponential. The rate constant is 0.0287 +/- 0.0022 s-1. Using ghosts with BSA, the kinetics is biexponential with widely different rate constants. Extrapolated zero-time values reflect, according to additional investigations, 'instantaneous' release of PA from the outer surface of the ghosts. Analyses of the biexponential curve up to about 55% tracer efflux assign unequivocally values to three model parameters. (1) k1, the dissociation rate constant of the PA-BSA complex is (1.47 +/- 0.03) x 10(-3) s-1 and (2.56 +/- 0.08) x 10(-3) s-1 and (4.08 +/- 0.13) x 10(-3) s-1 at v = 0.2, 0.6 and 1.4, respectively. (2) k3*, the overall rate constant of PA transport from the inside of the ghost membrane to the medium is 0.0269 +/- 0.0020 s-1 independent of v. (3) Qkin, the ratio of PA on the inside of the membrane to PA on BSA within the ghosts is v dependent and smaller than a corresponding ratio Qeq measured in equilibrium by a value corresponding to PA on the outer surface. This fraction is released with a rate constant, k5, which is of the order of 1 s-1. The data suggest a maximum PA transport capacity, Jmax, of 2 pmol min-1 cm-2, 0 degrees C, pH 7.3.     

Sheep Erythrocyte Membrane Binding and Transfer of Long-Chain Fatty Acids

We have studied binding and membrane transfer rates of unsaturated long-chain fatty acids in sheep red cells, as previously done for human red cells, in order to elucidate the transport mechanism. Observed differences must be assigned to the different composition of the membrane in the two species. Equal surface areas of the membranes of the two species have similar binding capacities and affinities for palmitic-, linoleic-, oleic- and arachidonic acid at 37°C. The competitive bindings of linoleic- and arachidonic acid as well as the distribution of bound arachidonic acid on the two sides of the membrane are not different in the two species. However, the rate constants for membrane transfer in sheep are less than half of those measured previously for human ghosts. This finding is confirmed by the exchange efflux kinetics of ghosts containing albumin-bound fatty acid. Studies of sheep ghost membranes with oleic-, arachidonic- and linoleic acid reveal a proportionality between the membrane transfer rate constants and the number of fatty acid double bonds, as found previously for human ghost membrane, and the effect of double bonds is in harmony with a large negative activation entropy for diffusion through the membrane. The established replacement of lecithin by sphingomyelin with a low unsaturation fatty acid index in sheep membranes probably causes a lower transversal lipid phase fluidity. Double bonds diminish the flexibility of hydrocarbon chains and thus the large negative activation entropy of diffusion across the membrane. The smaller transfer rate constants of the three unsaturated fatty acids in sheep membranes support the hypothesis that the transfer is diffusion in protein defined annular lipid domains and not carrier mediated. Received: 24 February 1999/Revised: 10 June 1999     

Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor

Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.     

Transmembrane linkage between surface glycoproteins and components of the cytoplasm in neutrophil leukocytes

An experimental approach is described that enables the analysis of interactions between exogenous surface ligands and components of the cytoplasm in neutrophil leukocytes. Neutrophils treated with the nonionic detergent Lubrol PX, under controlled conditions, yield intact detergent-insoluble ghosts. Morphological analysis of neutrophil ghosts shows that they retain the original dimensions of the cell and consist almost entirely of a peripheral filamentous network, representing the submembranous cortical web, concentric to nuclear remnants. All intracellular membrane-bounded organelles, plasma membrane, and background cytoplasmic electron density are absent. Biochemical analysis of the ghosts shows that less than 10% of enzyme markers for the soluble and granule fractions remain, and that greater than 90% of total cell phospholipid is removed during detergent extraction. The major proteins remaining in the ghosts comigrate, on polyacrylamide gels in the presence of SDS, with chicken gizzard actin, myosin, filamin, and a 110-kdalton protein. Patches and caps induced on neutrophils with either fluorescein isothiocyanate-concanavalin A or ferritin-concanavalin A retain their original location and morphology on ghosts after lysis, as determined by both fluorescence and electron microscopy. In similar experiments, but using 125I-labeled lectins, 37% of total cell bound concanavalin A (Con A) and 25% succinylated Con A remain attached to the ghosts. A major 125I-labeled membrane glycoprotein (80 kdaltons) is associated with ghosts prepared from intact neutrophils iodinated in the presence of exogenous lactoperoxidase. Further 125I-labeled membrane glycoproteins (217, 170, and 147 kdaltons) become associated with ghosts prepared from iodinated cells treated before lysis with Con A, but not with succinylated Con A. These data taken together suggest that linkages exist in neutrophils between proteins exposed on the outer surface of the plasma membrane and the peripheral filamentous network independent of the presence of lipid bilayer. The implications of these findings for surface motile phenomena will be discussed.