Reaction of ozone with phospholipid vesicles and human erythrocyte ghosts

Phospholipid vesicles (unilamellar) and liposomes (multilamellar) made from egg phosphatidylcholine reacted similarly with ozone, producing hydrogen peroxide and malonaldehyde. On the basis of amount of ozone reacted, there was a 20% yield of hydrogen peroxide and 2.4% yield of malonaldehyde. The reactivity of the egg phosphatidylcholine membranes was a function of exposed membrane surface area. Large amounts of ozone caused no change in erythrocyte ghost phospholipid, fatty acid, or cholesterol composition. Thiobarbituric acid-positive material and conjugated dienes were present in very small quantities, suggesting some lipid oxidation which was below the limits of chromatographic detection. Ozone inhibited glyceraldehyde 3-phosphate dehydrogenase more than (Na⁺ + K⁺) adenosine triphosphate in exposed unsealed erythrocyte ghosts. The (Na⁺ + K⁺) adenosine triphosphatase activity sensitive to ozone was the ouabain-insensitive activity. Acetylcholinesterase activity was not significantly inhibited.     

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.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes.

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest 125-I-labelled casein, covalently linked to latex beads, have been examined. Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8-9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade 125-I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Proteolytic activity associated with human erythrocyte membranes. Self-digestion of isolated human erythrocyte membranes

At least two kinds of enzymes are active in the proteolytic self-digestion of erythrocyte membranes. The specific activities of these enzymes do not decrease with repeated washings of purified stroma. The effects of a variety of inhibitors on the membrane preparation's capacity to digest ¹²⁵I-labelled casein, covalently linked to latex beads, have been examined.Pepstatin-inhibitable enzyme, active at low pH, digests the membrane extensively to small polypeptide fragments. Spectrin, located at the internal part of the membrane, is readily degraded. Diisopropylfluorophosphate-inhibitable enzyme, active at pH 8–9, has only limited digestive capacity. Some of the membrane components, such as the small molecular weight glycoproteins, are resistant to digestion. The restricted capacity of digestion is due to the membrane molecular arrangement; increased disaggregation removes the restriction and increases the activity. Spectrin is not digested unless the membrane topography is disrupted by NP-40 neutral detergent. These observations suggest that the enzymes active at basic pH are located external to the cell. Intact cells do possess a limited capacity to degrade ¹²⁵I-labelled casein when their surfaces are brought into contact with substrate-coated beads.     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

Proteolytic activity of human erythrocyte membrane during ghosts preparation

1. Proteins in human erythrocyte membranes after red blood cells hemolysis revealed relatively high rate of self-digestion. 2. This indicates hemolysis as a critical moment for membrane proteases activation. 3. The detailed pattern of band 3 protein and spectrin degradation during ghosts preparation was more complicated and reflected both the changes in proteolytic susceptibility and extraction of some proteases. 4. Further extraction of membrane proteins by alkali stripping resulted in an increase in the self-digestion rate and decrease in the degradation rate of an exogenous substrate.     

Ionophoric activity of truncated gramicidin A analogue in phospholipid bilayers and mitochondrial and erythrocyte membranes

Ionophoric activities of an N-terminus truncated gramicidin A (gA) analogue (mini-gramicidin) and its covalent dimer were studied in planar bilayer phospholipid membranes (BLM) using macroscopic current measurements (at high concentrations of the peptides) and single-channel recordings. As with gA-induced currents, mini-gramicidin-stimulated macroscopic currents through BLM underwent sensitized photoinactivation, i.e. were suppressed after irradiation with visible light in the presence of a photosensitizer generating singlet oxygen. The sensitivity of the tested compounds to photoinactivation descended in the following order: minigramicidin dimer > mini-gramicidin monomer > gA. The data from single-channel measurements and kinetic analysis of flash-induced photoinactivation obtained at different levels of macroscopic currents suggest that mini-gramicidin and its covalent dimer induce a variety of conducting states; their ratio depends on membrane thickness. Analysis of natural (mitochondrial and erythrocyte) membranes established that ionophoric activities of mini-gramicidin and its covalent dimer depend essentially on the membrane type.     

Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes.

Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.     

Rotational diffusion of band 3 in erythrocyte membranes. 1. Comparison of ghosts and intact cells.

The rotational diffusion of eosin-labeled 3 in human erythrocyte cells and hemoglobin-free ghosts at 37 degrees C has been studied in detail by polarized delayed luminescence. The time-resolved anisotropy with both cells and freshly prepared ghosts is similar, decaying with well-resolved rotational correlation times of 0.03, 0.2, and greater than or equal to 1 ms. Mild proteolytic removal of the water-soluble 41-kDa cytoplasmic domain of band 3 in ghosts results in a drastic increase in the fractional contributions of the two fastest depolarizing components. Our results, taken together with other data in the literature, imply that several classes of band 3 that differ greatly in mobility exist in ghosts and intact cells. The mobility of one class is hindered due to complexation with other membrane or cytoplasmic proteins mediated via the 41-kDa cytoplasmic domain. However, another class of band 3 molecules exists as homo-or heterooligomeric complexes larger than a dimer that are stabilized by hydrophobic interactions involving the intramembranal domain. Finally, the presence of the (previously undetected) 0.03-ms anisotropy component strongly suggests that a significant fraction of band 3 in both ghosts and intact cells is highly mobile and diffuses at the rate expected for a freely rotating dimer in the erythrocyte membrane.     

Functional and pathological significance of phospholipid asymmetry in erythrocyte membranes

The normal asymmetric distribution of phospholipids in the plasma membrane is perturbed in erythrocytes from patients with chronic myelogenous leukemia. Since experimentally-produced lipid-symmetric erythrocytes are more interactive with cells of the reticuloendothelial system than are their lipid-asymmetric counterparts, the biological recognition of chronic myelogenous leukemia erythrocytes by the reticuloendothelial system was examined. With one exception, all erythrocyte samples from patients with chronic/benign chronic myelogenous leukemia were more adherent to endothelial cells and more readily phagocytosed by macrophagesin vitro than were normal erythrocytes. Thus, these naturally occurring pathological erythrocytes display the same dysfunctional intercellular interactions as the laboratory models.     

Isolation of the terminal complement complex from target sheep erythrocyte membranes.

(1) Membranes from sheep erythrocytes lysed with antibody and human complement were solubilized in Triton X-100 and subjected to isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Membrane-bound serum proteins were located in the gels by subsequent immunoelectrophoresis against antisera to human serum proteins. Monospecific antisera against C9 and C5 were used to locate the terminal complement complex, which is not dissociated by Triton X-100. The complex focused between pH 5.8 and pH 6.5 and was separated from the bulk of other membrane-bound serum proteins, which focused at pH ranges below than 6.0. (2) In a second step, proteins electrophoretically eluted from the gel sections containing the terminal complement complex were chromatographed on Sepharose 6B equilibrated with 0.05% Triton X-100. Fused rocket immunoelectrophoresis was used to monitor separations. This step separated the terminal complement complex from the remaining contaminating proteins. The complex eluted in a broad peak corresponding to a molecular weight range of 800000-4000000. (3) The terminal complement complex thus obtained migrated with alpha-mobility and yielded a single precipitation arc in crossed immunoelectrophoresis using polyvalent antisera to human serum proteins. A distinct precipitate was obtained with monospecific anti-C9. The presence of C5 and C6, in complex with one another and with C9 was demonstrable by immuno-double-diffusion. No immunoprecipitate was obtained with antisera to sheep erythrocyte membrane proteins. (4) Dodecyl sulfate gel electrophoresis of the complex revealed seven protein bands of 190000, 160000, 115000, 93000, 85000, 68000 and 60000 daltons. Planimetric quantitation of densitometric scans gave a molar ratio of approx. 0.7:0.3:1:1:1:2:1 for these bands, respectively. All bands stained faintly with periodate-Schiff. Two-dimensional dodecyl sulfate gel electrophoresis showed that the first two bands (190000 and 160000 daltons, probably C5b and C5c) represented proteins possessing more than one peptide chain linked by disulfide bonds. The main subunit for both bands was a protein of approximately 68000 daltons. Band 5 (83000 daltons, probably C8alpha) was split into two peptide chains of approximately 68000 and 15000 daltons. The other components were not affected by dithiothreitol treatment. (5) The dodecyl sulfate gel electrophoretograms obtained were very similar to that described by Kolb and Müller-Eberhard (Kolb, W.P. and Müller-Eberhard, H.J. (1975) J. Exp. Med. 141, 724-735) for the terminal complement complex isolated from inulin-activated serum. However, certain minor but consistent deviations were observed. A preliminary correction of the electrophoretograms is presented.     

Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.     

Proteolytic activity in electropherograms (SDS-PAGE) of bovine erythrocyte ghosts

1. Activity of proteases, strongly related with erythrocyte membrane, was analysed employing a new methodological approach. 2. Intact bovine ghosts, ghosts depleted in peripheric proteins or purified Triton X-100 and ghost extracts were electrophoresed and the proteolytic activity in the gel fragments (SDS-PAGE) was assayed. 3. At least two proteases that were inhibited by EDTA and PMSF were found.     

Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.

1. The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2. Choline-containing phospholipids were found to comprise the majority of the outer layer of the membrane. 3. The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4. Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5. Indications were found for a rather slow flip-flop of lecithin across the membrane.