Calcium-dependent association of glutathione S-transferase with the human erythrocyte membrane

Elevations in intracellular calcium increase the adsorption of a cytoplasmic protein to human red blood cell membrane. This protein migrates on SDS polyacrylamide gels at 23,000 daltons and has been called band 8. The association of this protein with the membrane is increased in sickle cell anemia. This protein is extracted from the membrane with EGTA, a calcium chelator. Enzymatic and immunological studies identify band 8 as a glutathione S-transferase.     

The partial amino acid sequence of human erythrocyte catalase

More than 95% of the sequence of human erythrocyte catalase (HEC) has been determined. There are at least 41 differences in sequence between it and that of bovine liver and erythrocyte catalases (BLC and BEC). Although the normal subunit length of BLC is 506 residues, BEC has at least 517 and HEC 520 residues. Most differences between HEC and BLC or BEC are conservative substitutions. If the heme-protein contacts as determined by X-ray crystallography are examined, only one of 39 residues in contact with the heme group differs between HEC and BLC or BEC.     

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.     

The kinetics of cyanide binding by human erythrocyte catalase

The kinetics of the binding reaction of cyanide by human erythrocyte catalase at 25 °C has been studied over the pH range 4.2 to 10.2 by means of temperature jump and stopped flow techniques. Catalase reacts with cyanide at a constant rate in the range pH 4.2 to 8.1 which decreases at higher pH. This is most simply explained by the reaction of catalase with unionized hydrogen cyanide molecules. The pH-independent rate constant for the formation of the catalase-cyanide complex is (1.3 ± 0.1) × 10⁶m^?1 s^?1. The association equilibrium constant and the dissociation rate constant for the catalase-cyanide complex were determined from the relaxation amplitudes of temperature jump experiments and by spectrophotometric titration and are (3.1 ± 0.2) × 10⁵m^?1 and 4.2 ± 0.6 s^?1, respectively in the pH-independent region.     

Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane

Binding of F-actin to spectrin-actin-depleted erythrocyte membrane inside-out vesicles was measured using [3H]F-actin. F-actin binding to vesicles at 25 degrees C was stimulated 5-10 fold by addition of spectrin dimers or tetramers to vesicles. Spectrin tetramer was twice as effective as dimer in stimulating actin binding, but neither tetramer nor dimer stimulated binding at 4 degrees C. The addition of purified erythrocyte membrane protein band 4.1 to spectrin- reconstituted vesicles doubled their actin-binding capacity. Trypsinization of unreconstituted vesicles that contain < 10% of the spectrin but nearly all of the band 4.1, relative to ghosts, decreased their F-actin-binding capacity by 70%. Whereas little or none of the residual spectrin was affected by trypsinization, band 4.1 was significantly degraded. Our results show that spectrin can anchor actin filaments to the cytoplasmic surface of erythrocyte membranes and suggest that band 4.1 may be importantly involved in the association.     

Spectral studies of human erythrocyte catalase.

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as alpha helix, beta pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.     

The anticancer drug cisplatin interacts with the human erythrocyte membrane

Drugs which exert their effects by interacting with DNA cause structural and functional membrane alterations which may be essential for growth inhibition by these agents. This paper describes the interaction of cisplatin with the human erythrocyte membrane and models constituted by bilayers of dimyristoylphosphatidylethanolamine (DMPE) and diacylphosphatidylserine (DAPS), representative of phospholipid classes located in the inner monolayer of the erythrocyte membrane, and of dimyristoylphosphatidylcholine (DMPC), a class present in its outer monolayer. Cisplatin ability to perturb DMPE, DAPS and DMPC bilayer structures was determined by X-ray diffraction and fluorescence spectroscopy. Electron microscopy disclosed that human erythrocytes incubated with 35 microM cisplatin, which is its therapeutical concentration in serum, developed cup-shaped forms (stomatocytes). According to the bilayer couple hypothesis, this means that the drug is inserted into the inner monolayer of the erythrocyte membrane, a conclusion supported by the studies on model systems.     

Interaction of chlorpromazine with the human erythrocyte membrane

The interaction of the amphipath chlorpromazine (CPZ) with the human erythrocyte membrane was evaluated. The partition coefficient of CPZ between the membrane bilayer and the aqueous compartment, measured spectrophotometrically, ranged between 1 and 3 X 10(3). An independent estimate, 4.6 X 10(3), was obtained by a novel method which avoided the measurement of binding and determined instead the variation of the hemolytic potency of the amphipath with the ratio of buffer volume to membrane volume. The maximal uptake of CPZ exceeded 2 X 10(9) molecules/red cell, corresponding to a volume greater than that of the bilayer itself. Such heavily loaded membranes were increased in thickness more than 2-fold, suggesting the formation of a CPZ-rich zone at the center of the bilayer. Ghosts loaded with massive levels of CPZ condensed approximately 20-fold in surface area and increased proportionately in thickness, suggesting the formation of a novel CPZ-lipid solution. CPZ caused hemolysis by a colloid-osmotic mechanism. By measuring the simultaneous uptake of mannitol and sucrose, we determined that CPZ induced holes of constant size but variable number. If circular, the holes would have had a diameter of approximately 14 A. The time-averaged number of holes ranged from 0.09 per cell (signifying intermittency) to 16. Freeze-fracture electron microscopy of CPZ-treated red cells revealed multiple round patches of nearly particle-free bilayer up to 0.3 micron in diameter with crowding of the intramembrane particles into the surrounding membrane. We interpret these images to signify lateral phase separation within the CPZ-treated bilayer. Hemolysis could, therefore, result from the intermittent opening of weak seams at phase boundaries; these could then be fluctuating slits approximately 14 A in width and of variable length, rather than simple circular holes.     

The exchangeability of human erythrocyte membrane cholesterol

A new method has been used to determine what fraction of human erythrocyte cholesterol is available for exchange with plasma unesterified cholesterol. Erythrocytes labeled with ³H-cholesterol by this exchange process were incubated with sonicated phosphatidylcholine vesicles, giving rise to a net movement of cholesterol out of the cells. The specific activity of cholesterol taken up by the vesicles depended on the length of time of incubation. Initially the specific activity in the vesicles was greater than that in the cells, but after approximately 10% of cell cholesterol had been removed, the specific activity of subsequently removed cholesterol was equal to that of the remaining erythrocyte cholesterol. We conclude from these data that (a) all of the cholesterol in the erythrocyte is exchangeable with plasma, and (b) approximately 10% of erythrocyte cholesterol is in a more rapidly exchangeable pool than the remainder.     

Human and mouse hemoglobin association with the transgenic mouse erythrocyte membrane

Transgenic mouse models of hemoglobinopathies unravel pathophysiological mechanisms; yet the validity of the red blood cell (RBC) model of human hemoglobin (hHb) enveloped by a mouse (m) membrane has been questioned. Isoelectric focusing of hHb and mHb from transgenic mRBC shows a greater association of mHb to the mouse membrane compared to normal hHbA, supporting a species-specific Hb-mRBC membrane interaction. Enhanced hmutant Hb (HbE, HbS and HbC)-mRBC membrane affinities correlates with enhanced membrane lipid peroxidation and parallel those reported in hRBC, lending support to transgenic mRBC as models of hemoglobinopathies. Species-specific Hb-membrane interaction may be overridden by Hb charge and conformational alterations.     

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1605-1618).     

The reactivity of human erythrocyte membrane cholesterol with a cholesterol oxidase

Cholesterol oxidase (EC 1.1.3.6, Brevibacterium sp.), which catalyzes the reaction: cholesterol + O₂ → Δ4-cholestenone + H₂O₂, has no effect on the cholesterol of intact (human) erythrocytes and of “resealed” ghosts, when it is present only outside these ghosts. The cholesterol of “leaky” ghosts, of “resealed” ghosts with enzyme trapped within, and of “inside-out” vesicles, was completely oxidized. This pattern indicates that the inner (cytoplasmic) membrane surface must be exposed to the enzyme for the reaction to occur, and that outer surface cholesterol only becomes reactive after the membrane has been degraded by the oxidation of inner surface cholesterol. The enzymatic oxidations followed monotonic first-order kinetics, and hence gave no evidence to support the two states of cholesterol in the membrane that had been postulated earlier from studies on the plasma lipoprotein extraction of cholesterol from the membrane.     

The anticancer drug cytarabine does not interact with the human erythrocyte membrane

Cytarabine, an analog of deoxycytidine, is an important agent in the treatment of ovarian carcinoma, acute myeloid and lymphoblastic leukemia. Its mechanism of action has been attributed to an interference with DNA replication. The plasma membrane has received increasing attention as a possible target of antitumor drugs, where the drugs may act as growth factor antagonists and receptor blockers, interfere with mitogenic signal transduction or exert direct cytotoxic effects. Furthermore, it has been reported that drugs that exert their antiproliferative effect by interacting with DNA generally cause structural and functional membrane alterations which may be essential for growth inhibition by these agents. This paper describes the studies undertaken to determine the structural effects induced by cytarabine to cell membranes. The results showed that cytarabine, at a concentration about one thousand times higher than that found in plasma when it is therapeutically administered, did not induce significant structural perturbation in any of these systems. Therefore, it can be unambiguously concluded that this widely used anticancer drug does not interact at all with erythrocyte membranes.     

Centrifugal analysis of undiluted packed human erythrocyte lysates studies of the association of glyceraldehyde-phosphate dehydrogenase with the membrane fraction

Concentrated human erythroyte lysates (greater than 99% initial haematocrit) were subjected to high centrifugal fields. This caused the membrane fraction to separate from the cytoplasmic portion, due to the lower density of the former. Enzyme distribution data indicated that glyceraldehyde-phosphate dehydrogenase was predominantly in the cytoplasmic fraction.     

The transport of univalent thallium across the human erythrocyte membrane

Transport of 204Tl was studied in human erythrocytes incubated in isotonic salt solutions at pH 7.4 and 37 degrees C. 204Tl was rapidly accumulated in cells up to the constant level within a 10 minutes incubation (t0.5 = 3.5 min). The rate of uptake and the distribution ratio decreased in the presence of 0.1 mM ouabain and 0.5-1.0 mM furosemide (t0.5 = 5 min). A broad variability of the coefficient 204Tl distribution was observed in the intact erythrocytes due to a ouabain-sensitive component which was seen to decrease with the increase in Tl+ concentration in the medium (0.005-0.2 mM), and also to depend on the medium ion composition. On the contrary, a passive distribution of 204Tl in the presence of ouabain and furosemide was relatively constant within 1.1-1.5. The steady state distribution of 204Tl was declined after a substitution of Cl- by sucrose in the medium due to depolarization of erythrocyte membrane. On the other side, 204Tl uptake by the cells was raised during hyperpolarization of the membrane in the presence of valinomycin.     

The diversity of sulfhydryl groups in the human erythrocyte membrane

Human bank blood erythrocytes were exposed to the mercurials p-chloromercuribenzoate (PCMB), chlormerodrin (CM), p-chloromercuribenzenesulfonate (PCMBS), and 1-bromomercuri-2-hydroxypropane (BMHP) for different time intervals, at different concentrations and in combination with n-ethylmaleimide (NEM) added before, and 2-mercaptoethylguanidine (MEG) and reduced glutathione (GSH) added after the mercurial. Binding patterns of the mercurials to the cells and effects on permeability of the cells were measured. The results indicate that the erythrocyte membrane contains multiple classes of sulfhydryl groups, alteration of which has a variety of effects on cell permeability. PCMB, chlormerodrin and PCMBS react with at least three classes of sulfhydryls, two of which are associated with the sodium-potassium barrier and, when altered, result in potassium loss, sodium accumulation and hemolysis. BMHP reacts with at least two classes of sulfhydryls, one of which is associated with permeability, and, when altered, results in hemolysis in isotonic solutions of choline chloride or lactose. The results provide additional insight into the structure and function of the erythrocyte membrane.