Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.     

Permeability properties of erythrocyte ghosts

1. Erythrocyte ghosts from human blood were produced by gentle water hemolysis. The ghost-containing hemolysate (about 20 mN) was added to media of different composition (KCl, NaCl, glucose, sucrose, etc.) and varying concentration ranging from 8 to 840 mN. The volume changes of the ghost cells were followed by a light absorption method. The potassium and sodium concentrations were also analyzed in some representative cases. 2. The ghosts shrank, or swelled, in two stages. An initial phase with a momentary expulsion, or uptake, of water leading to an osmotic equilibrium, was followed by a second phase in which a slow swelling or shrinking proceeded toward a final constant volume. 3. The ghosts were semipermeable in the sense that water always passed rapidly in either direction so as to maintain isotonicity with the external medium. The relation between ghost cell volumes (V) and the total concentration (C(e)) of the suspension medium can be expressed by a modified van't Hoff-Mariotte law: (C(e) + a)(V - b) = constant. Here a is a term correcting for an internal pressure and b is the non-solvent volume of the ghost cells. This means that the ghosts behave as perfect osmometers. 4. On the other hand appreciable concentration differences of the K and Na ions could be maintained across the intact ghost cell membranes for long periods. Whether this phenomenon is due simply to very low cation permeability or to active transport processes cannot be decided, although the first assumption appears more probable. 5. When the ghosts were treated with small concentrations of a lytic substance like Na oleate, the alkali ion transfer was greatly increased. This seems to be a simple exchange diffusion process with simultaneous, continued maintenance of osmotic equilibrium (= the second phase). A simplified theory is also given for the kinetics of the volume variations and ion exchange during the second phase (cf. the Appendix). 6. Miscellaneous observations on the effects of pH, and of some other substances are discussed. Some shape transformations of the ghost cells are also described.     

Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts.     

Outer cell envelope membrane and shape of Spirillum serpens.

“Ghosts” have been isolated from Spirillum serpens that are free of murein, are surrounded by a unit membrane (derived from the outer membrane of the cell envelope), have lost all intracellular material (except for some poly-β-hydroxybutyrate), and still maintain Spirillum's shape.The ghost membrane contains about 50% protein which is resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three bands corresponding to apparent molecular weights between 21,000 and 40,000, and the major protein band I (40,000) consists of at least two (Ia and Ib) but not many more polypeptide chains. HP-layer protein (hexagonally packed surface protein) is absent. At least one of the latter polypeptide chains is required for the establishment of the long-range order apparent in ghosts since proteases degrade band I proteins and concomitantly destroy the ghost. The other polypeptides (II and III) do not appear to be required for maintenance of shape of the Spirillum ghost since their amounts can vary widely from preparation to preparation. Ghosts as well as cells can be cross-linked with dimethyl diimidoesters. Such ghosts proved to be cross-linked over their entire surface, and a covalently closed macromolecule of the size of the cell had been created. Under certain conditions of cross-linking these ghosts upon extraction with hot sodium dodecyl sulphate were pure protein. Ammonolysis of this material liberated band I protein.These findings strongly suggest that there is a rather dense packing of the protein in the ghost membrane, and proteins Ia and Ib may be arranged as repeating subunits in the sense that protein-protein interaction exists along the whole membrane. Several observations also suggest that the ghost membrane concerning the arrangement of these proteins does not represent a gross artifact regarding the outer cell envelope membrane. The possibility exists that the assembly of polypeptides Ia and Ib participates in the determination of cellular shape.     

Mobilization of iron from specifically labeled reticulocyte ghosts.

Specifically labeled 59Fe ghosts have been prepared by incubation of whole reticulocytes with 59Fe3+-transferrin-CO3(2)-- followed by washing and ghost isolation. The binding of 59Fe by the membrane fraction is quite stable over a wide range of conditions, but iron mobilization occurs on incubation with chelating agents or cell lysate. The time course of 59Fe mobilization by unlabeled reticulocyte lysate exhibits five apparently zero-order phases. The rate of iron mobilization is linearly dependent on the concentration of 59Fe ghosts present in the incubation mixture. In contrast, the relative concentration of lysate appears to exhibit a saturation dependence with regard to membrane iron mobilization. Bathophenanthroline sulfonate follows a multiphasic time course of iron mobilization similar to that found with the lysate. Lysate from mature erythrocytes was found to mobilize iron with kinetics that are identical to reticulocyte lysate. The number and duration of the phases is independent of the mobilizing agent. The role of the membrane fraction in regulating the rate of iron release to cytosol was also investigated by the repetitive incubation of 59Fe ghosts with fresh lysate. The rate of 59Fe mobilization depended on the condition of the ghost with regard to prior 59Fe depletion. This publication emphasizes the active role of the membrane fraction in determining the rate at which iron will become available to the cytosol and the possibility that cytosol factors modulate the action of membrane bound components.     

Trypsin-induced changes in the orientation of latent ATPase in protoplast ghosts from Mycobacterium phlei.

Latent ATPase, located on the inner surface of protoplast ghosts of Mycobacterium phlei, was unmasked either by trypsin or an impermeable form of trypsin, ethylene maleic anhydride-trypsin. Density gradient experiments showed that the ghost preparations remained intact following trypsin treatment. Evidence was obtained that 125I-trypsin failed to penetrate the ghost membranes. Thus, attempts were made to determine whether the ATPase molecule in the ghost membranes is accessible from the outer surface. Treatment of protoplast ghosts and trypsin-treated ghosts with 125I by the lactoperoxidase method resulted in the labeling of ATPase only in the trypsin-treated ghost preparations. The antibody to latent ATPase inhibited ATPase activity in trypsin-treated ghosts. The changes in the fluorescence polarization of diphenyl hexatriene indicated that trypsin treatment of the ghost membranes resulted in an increase in membrane fluidity. These studies suggest that the latent ATPase moiety has undergone translocation to the outer surface or it became accessible to trypsin digestion from the outer surface of the membranes as a result of removal of some proteins covering ATPase molecule in the membranes.     

Fluidity of human erythrocyte ghosts.

The fluidity, defined by its two components, the order parameter, S, and the rotation correlation time, tau c, was studied on healthy human erythrocytes ghosts. We also measured ghost protein, cholesterol and phospholipid contents as well as acetylcholinesterase activities. No statistically significant difference was evidenced between erythrocyte ghosts from men and women. Whereas tau c values did not significantly vary among sample elements, variations of ghost order parameters about the mean were explained at 61% by changes in cholesterol contents and, to a lesser extent, in protein contents. No relationship was evidenced between ghost order parameter values and those of corresponding acetylcholinesterase activities. Liposomes prepared from ghost lipid extracts had much lower order parameter values than did corresponding ghosts. A few experiments were performed in the same way on ghosts from sickle blood. This disease appeared to decrease the bilayer lipid motionnal freedom as an increase of the order parameter values was evidenced.     

Water-phase palmitate concentrations in equilibrium with albumin-bound palmitate in a biological system.

The palmitate (PA) binding and transport capacity of human and bovine red cell membranes enables us to establish, in a biological system, the existence of a well-defined monomer concentration in equilibrium with PA bound to bovine serum albumin (BSA, 30 microM) inside the resealed red cell ghosts. Supernatants of suspensions of the [3H]PA-labeled ghosts contain a tiny quantity of dissolved binding capacities besides the monomer PA. This is demonstrated by linear regression of supernatant tracer concentrations versus ghost concentrations in a dilution series. The extrapolated value, corresponding to zero ghost concentration, is the monomer PA concentration in equilibrium with PA bound to BSA within the ghosts in molar ratio (nu). Measurements have been carried out for nu between 0.1 and 1.5 and at 0 degrees C, 10 degrees C, 23 degrees C and 38 degrees C. The important nu-dependent binding of PA to the ghost membrane itself enables us to use preparations of BSA-free ghosts in the same way, whereas this is impossible in the case of arachidonic acid. Within the physiological range of nu the PA monomer concentrations are accounted for by an apparent dissociation equilibrium constant (Kd) 3.4 10(-8) M at 38 degrees C calculated on basis of three equivalent binding sites per mol BSA. Kd depends on temperature with a well-defined enthalpy of 38.4 kJ/mol.     

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton.

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.     

Regulatory Properties of Acetokinase from Veillonella alcalescens

Ghosts of T4 bacteriophage inhibit the uptake of thiomethyl-beta-galactoside (TMG), alpha-methylglucoside, glucose-6-phosphate, and glycerol in Escherichia coli B. The transport of orthonitrophenyl-beta-galactoside (ONPG) is also inhibited to a lesser degree and without alteration of the apparent K(m) of transport. These effects of ghosts parallel those of energy poisons on these systems. However, no one energy poison can produce such pronounced inhibitory effects in all these systems. The effect of the intact phage in these systems was either absent or very slight relative to the ghost. The effect of ghosts on the uptake of TMG was not immediate; at 10 C, no effect of the ghosts was apparent for at least 2 min. This suggests that a step, more temperature dependent than the attachment of the ghost, is necessary for the inhibitory action. The intracellular level of adenosine triphosphate (ATP) in the ghost-infected cells fell to less than 25% of the control value, and the ATP lost from the cell appeared in extracellular medium. Phage, on the other hand, caused no decrease in the intracellular ATP level. This loss of ATP from the cells after ghost infection suggests an alteration of the barrier properties of the membrane so that ATP can leave the cell; however, the accessibility of extracellular ONPG to intracellular beta-galactosidase does not increase. The dissimilarity of the actions of phage and ghosts on all properties examined does not support the model that the initial events in their infections are identical but that the intact phage, unlike the ghost, can provide information for the repair of its effects.     

Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation.

Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis.

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts. Two-dimensional patterns of ghost proteins were not heavily contaminated (less than or equal to 25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8--10 fold over whole cells. Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Extended recombinant bacterial ghost system.

Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines. In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences. Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens. In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis. Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes. As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines. Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts. Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production. This fact explains the superior quality of ghosts when compared to other inactivated vaccines. The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines. As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized. This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.     

In situ solid-state NMR study of antimicrobial peptide interactions with erythrocyte membranes

Antimicrobial peptides are promising therapeutic agents to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane and are thus less likely to induce resistance. Because they are membrane-active molecules, it is critical to verify and understand their potential action toward eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ ³¹P and ²H solid-state NMR (SS-NMR). We established a protocol to integrate up to 25% of deuterated fatty acids in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. Fatty acid incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the antimicrobial peptides. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by ³¹P and ²H SS-NMR is consistent with membrane perturbation through a carpet mechanism for aurein 1.2, whereas caerin 1.1 acts on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, highlighting the importance of hydrophobicity when determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity toward eukaryotic cells.     

Phospholipase activity in bacteriophage-infected Escherichia. II. Activation of phospholipase by T4 ghost infection.

The release of free fatty acids from the phospholipids of Escherichia coli is initiated immediately after the attachment of T4 ghosts. A similar accumulation of free fatty acids is observed if the cells are infected with T4 phage in the presence of chloramphenicol or puromycin. An early accumulation of free fatty acids, however, is not observed in T4 infections in which chloramphenicol or puromycin are not present, nor does it occur if the E. coli are infected with T4 phage before ghost infection, suggesting that phage products can prevent the phospholipid deacylation. If E. coli is infected with T4 ghosts before T4 phage infection, the accumulation of free fatty acids is not suppressed. When phospholipase-deficient E, coli are infected with T4 ghosts the appearance of free fatty acids is not observed, suggesting that T4 ghost attachment can activate the phospholipase of wild-type E. coli. Although the formation of free fatty acid apparently is a consequence of activation of the detergent-resistant phospholipase of the outer membrane, it is not observed in mutants deficient in the detergent-sensitive phospholipase.     

Yeast plasma membrane ghosts. An analysis of proteins by two-dimensional gel electrophoresis

We have examined yeast cell ghost preparations to assess their value in obtaining plasma membrane proteins. Ghosts prepared by two methods involving stabilization of spheroplast envelopes had similar protein patterns by two-dimensional gel electrophoresis, and approximately 200 proteins were resolved. Spheroplasts were lactoperoxidase iodinated, and recovery of label in ghost preparations was greater than 60%. Spheroplasts appeared to be impermeable to the lactoperoxidase reagents as judged by an examination of two-dimensional gel electrophoretic patterns of ghost proteins that had been iodinated in spheroplasts or in unsealed ghosts. Spheroplasts were also impermeable to pronase proteases. Surface iodination and surface proteolysis allowed us to identify exposed ghost proteins; the major ghost glycoprotein was exposed in spheroplasts.Two-dimensional patterns of ghost proteins were not heavily contaminated (?25% of all proteins) by proteins present in soluble or promitochondrial fractions, and estimates of surface label and total cell protein recovery suggested that the ghost fraction represents a cell envelope enrichment of 8–10 fold over whole cells.Resolution of ghost proteins by two-dimensional gel electrophoresis appears to be a powerful aid toward identifying membrane proteins.     

Structural and biochemical examination of ghosts derived from a deep rough (heptose-deficient lipopolysaccharide) strain and a smooth strain of Escherichia coli.

Outer membrane derived 'ghosts' can be readily generated from both smooth and deep rough (heptose-deficient LPS) strains of Escherichia coli 08. MORPHOlogical and biochemical studies confirmed that 'ghosts' of both strains are composed of protein (four major proteins), LPS, and phospholipid (cardiolipin and phosphatidylethanolamine) in the form of a single membrane of roughly the same shape as intact normal cells. The ghost membrane cleaves only slightly in freeze-etch preparations of ghosts derived from the smooth strain as compared to the extensive cleavage plane of ghosts derived from the rough strain. The asymmetrical distribution of ghost proteins was visualized, by critical point drying and shadowing with platinum, as a relatively smooth outer surface with some discernible particles (10-15 nm) and an extremely particulate inner surface (10-15-mm particles. Ghosts derived from the smooth strain retained their structure following chloroform-methanol extraction, while ghosts derived from the rough strain fragmented with chloroform-methanol extraction. Evidence is presented that LPS-protein interactions as well as protein-protein interactions are significant in maintaining the ghost structure.     

Membrane transport of L-triiodthyronine by human red cell ghosts.

L-T₃ transport has been investigated in human red cell ghosts. Determination of initial T₃ uptake revealed two separate saturable uptake systems, one with a K_m of 1.6 × 10^?8M, the other with a K_m of 3.3 × 10^?6M. Binding experiments resulted in two dissociation constants, 1.4 × 10^?7M.and 2.6 × 10^?6M. Uptake was dependent on the ghost volume, indicating an intravesicular location of T₃. The T₃ was concentrated 6 times by the ghosts. Ouabain reduced the uptake by the low K_m system, but was without effect on the high K_m system. Thus evidence is provided both of binding of T₃ to the ghost membrane and of its uphill transport across the membrane.     

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.