Protein and protein-lipid membranes from solubilized erythrocyte ghosts

Summary Erythrocyte ghosts were solubilized by addition of acid 2-chloroethanol to an aqueous membrane suspension. The proteins were separated from the lipids by chromatography on Sephadex LH-20 (Zahler and Wallach, 1967). Both the solution of separated proteins and of the total membrane in chloroethanol-water can be spread at a benzene-water interface. By lowering a thin teflon plate with a small hole through this interface, one can form protein or protein-lipid films over the hole. After the benzene has evaporated stable thin membranes are formed which contain only protein or protein together with lipid. The morphology and thickness of these membranes were investigated by different electron microscopic techniques.     

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts

Summary A model for the binding of 5-nitroxide stearate, I(12,3). to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L<1/2500), I(12,3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L=1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (>1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12,3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both dilute and clustered I(12,3) are due to membrane-incorporated probe.     

Amiloride fluxes across erythrocyte membranes

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.     

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.     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

The proton gradient across mycoplasma membranes

The proton gradient across mycoplasma membranes was determined by using different probes which distribute between the intracellular space and the suspension medium in response to a transmembrane proton gradient. The intracellular pH of intact glycolyzing mycoplasmas was generally more alkaline than the extracellular medium: pH_ext=7 and pH_int=7.4; hence, pH=0.4. The size of the proton gradient depended upon the extracellular pH. Without nutrient substrate, the mycoplasmas were unable to maintain a transmembrane proton gradient, i.e., pH approximated O.N, N-dicyclohexylcarbodiimide, an inhibitor of membrane-bound ATPase, carbonyl cyanide-m-chlorophenyl hydrazone, a proton conductor, and gramicidin, an antibiotic forming cation conduction channels across membranes, strongly affected and even abolished the proton gradient across mycoplasma membranes. These substances also impaired the metabolic activity and viability of mycoplasmas.     

Osmotic water permeabilities of human placental microvillous and basal membranes

Summary Literature data suggest that water accumulation by the human fetus is driven by osmotic gradients of small solutes. However, the existence of such gradients has not been supported by prior measurements. Attempts to estimate the size of the gradient necessary to drive net water movement have been seriously hampered by the lack of permeability data for the syncytiotrophoblast membranes. Stopped-flow light scattering techniques were employed to measure the osmotic water permeability (P _f )of microvillous (MVM) and basal membrane (BM) vesicles isolated from human term placenta. At 37°C, the P _f was determined to be 1.9±0.06 × 10^+–3 cm/sec for MVM and 3.1±0.20 × 10^+–3 cm/sec for BM (mean ±SD, n = 6). At 23°C, P _f was reduced to 0.7±0.04 × 10^+–3 cm/sec in MVM and 1.6±0.05 × 10^+–3 cm/sec in BM. These P _f values are comparable to those observed in membranes where water has been shown to permeate via a lipid diffusive mechanism. Arrhenius plots of P _f over the range 20–40°C were linear, with activation energies of 13.6 ± 0.6 kcal/mol for MVM and 12.9±1.0 kcal/mol for BM. Water permeation was not affected by mercurial sulfhydryl agents and glucose transport inhibitors. These data clearly suggest that water movement across human syncytiotrophoblast membranes occurs by a lipid diffusion pathway. As noted in several other epithelial tissues, the basal membrane has a higher water permeability than the microvillous membrane. It is speculated that water accumulation by the human fetus could be driven by a solute gradient small enough to be within the error of osmolarity measurements.We thank the staff of the labor and delivery ward at University of San Francisco Medical Center for help in obtaining placental tissue. This work was supported by NIH grant HD 26392. Dr. Jansson was supported by the Sweden-America Foundation, The Swedish Society of Medicine, The Swedish Society for Medical Research, and the Swedish Medical Research Council.     

A rapid method for the evaluation of the ionic permeabilities across epithelial cell membranes

This short note presents a recipe for the calculation of the ionic permeabilities across epithelial cell membranes. The method requires the Goldman-Hodgkin-Katz formalism as well as the consideration of the equivalent electrical circuit for an epithelial cell. The equivalent electrical circuit is solved in terms of the equivalent electromotive forces coupled in series with the ionic resistances of both cell membranes (apical and basolateral). The present procedure is feasible for any leaky epithelial cell membrane with the condition that this membrane (apical or basolateral) does not contain primary or secondary mechanisms for active transport.     

Structural research on the membranes and adjacent layers of erythrocytes during the production of erythrocyte ghosts

Using the polarization microscopy and X-ray crystal analysis it has been shown that the ordered structure is destroyed in membranes and adjacent layers of erythrocytes in the course of erythrocyte ghost preparation. These changes are accompanied by quenching of the birefringence which completely disappears in erythrocytes ghosts. The intensities of reflections in X-ray patterns are essentially decreased and the interlayer distances are changed.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

Thermotropic lipid phase separations in human erythrocyte ghosts and cholesterol-enriched rat liver plasma membranes

Summary Electron spin resonance (ESR) studies of human erythrocyte ghosts labeled with 5-nitroxide stearate, I(12,3), indicate that a temperature-dependent lipid phase separation occurs with a high onset at 38°C. Cooling below 38°C induces I(12,3) clustering. Similar phase separations were previously identified in human platelet and cholesterol-loaded [cholesterol/phospholipid molar ratio (C/P)=0.85] rat liver plasma membranes [L.M. Gordon et al., 1983;J. Membrane Biol. 76; 139–149]; these were attributed to redistribution of endogenous lipid components such that I(12,3) is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains. Further enrichment of rat liver plasma membranes to C/P ratios of 0.94–0.98 creates an artificial system equivalent to human erythrocyte ghosts (C/P=0.90), using such criteria as probe flexibility, temperature dependent I(12,3) clustering; and polarity of the probe environment. Consequently, cholesterol-rich and-poor domains probably exist in both erythrocyte ghosts and high cholesterol liver membranes at physiologic temperatures. The temperature dependence of cold-induced hypertonic lysis of intact human erythrocytes was examined by incubating cells in 0.9m sucrose for 10 min at 1°C intervals between 9 and 46°C (Stage 1), and then subjecting them to 0°C for 10 min (Stage 2). Plots of released hemoglobin are approx. sigmoidal, with no lysis below 18°C and maximal lysis above 40°C. The protective effect of low temperatures during Stage 1 may be due to the formation of cholesterol-rich domains that alter the bilayer distribution and/or conformation of critical membrane-associated proteins.     

Fluidity of erythrocyte membranes from patients with Duchenne muscular dystrophy: studies in intact erythrocytes and in ghosts

We have studied the alterations of fluidity in intact erythrocytes and in erythrocyte membranes from patients with Duchenne muscular dystrophy. The interest of this study was to comparison directly two types of results; these demonstrate an increase of fluidity in the erythrocytic membranes, no changes are present when the label is incorporated in intact erythrocytes. It might be inferred that hypotonic haemolysis removes components that are more weakly bound in Duchenne membranes, and that exert an immobilizing effect on the membrane lipids.     

ATP-driven proton fluxes across membranes of secretory organelles

The ATP-dependent proton uptake by chromaffin granule membranes, lysosomes, and synaptosomes was examined. In synaptosomes the reaction was absolutely dependent on the presence of chloride, while in chromaffin granules chloride had a profound effect and in lysosomes only a minor effect. The presence of chloride markedly increases the rate of collapse of delta pH by carbonyl cyanide p-trifluoromethoxyphenylhydrazone in all three organelles. Ascorbate with phenazine methosulfate uncoupled the ATP-dependent proton uptake by chromaffin granules, but had no effect on lysosomes and synaptosomes. Proton uptake by submitochondrial particles was about 50-fold more sensitive to dicyclohexylcarbodiimide than the proton uptake by chromaffin granule membranes. Chromaffin granule membranes were treated with 2 M sodium bromide to inactivate the mitochondrial ATPase. The treatment caused a complete inhibition of the ATP-dependent proton uptake. Solubilization of these membranes by sodium cholate, followed by reconstitution by cholate dilution revealed the ATP-dependent proton uptake of the system. It is concluded that the genuine ATPase enzyme of chromaffin granules is a proton translocator.     

Comparative aspects of phosphate transfer across mammalian erythrocyte membranes

Summary Magnitude and characteristics of phosphate transfer through the erythrocyte membranes of ten mammalian species were measured using tracer exchange techniques. Remarkable quantitative species differences could be demonstrated, permeabilities (at an extracellular phosphate concentration of 10mM) increasing from 0.2×10^–8 cm/sec (sheep) to 2.2×10^–8 cm/sec (rabbit) in the sequence sheep 相似文献   

Lipid photooxidation in erythrocyte ghosts: sensitization of the membranes toward ascorbate- and superoxide-induced peroxidation and lysis

The damaging effects of ascorbate (AH-) and superoxide (O-2) on resealed erythrocyte ghosts containing predetermined levels of lipid hydroperoxides (LOOHs) have been studied. Continuous blue light irradiation of membranes in the presence of protoporphyrin resulted in steadily increasing LOOH levels and enhanced release of a trapped marker, glucose 6-phosphate (G6P), after a 3- to 4-h lag. Neither superoxide dismutase (SOD) nor catalase inhibited these effects, ruling out O-2 and H2O2 as reactive intermediates. A 1-h light dose produced partially photoperoxidized ghosts, which, in the dark at 37 degrees C, released G6P no faster than unirradiated controls (approximately 7%/h). When xanthine oxidase plus xanthine (XO/X) was introduced as a source of O-2 and H2O2, the irradiated membranes lysed rapidly (t1/2 approximately 2 h). EDTA or SOD inhibited the reaction, whereas catalase had little or no effect. Unirradiated ghosts were not lysed by XO/X unless the system was supplemented with Fe(III), in which case total protection was afforded by SOD or catalase. In all experiments there was an excellent correlation between postirradiation lipid peroxidation (thiobarbituric acid reactivity) and G6P release. Similar observations were made with AH-. For example, dark incubation of photooxidized ghosts in the presence of 0.5 mM AH- resulted in rapid lysis (t1/2 approximately 1 h), which was stimulated approximately twofold by 50 microM Fe(III) and was inhibited by EDTA. By comparison, unirradiated ghosts showed no net peroxidation or lysis after 3 h exposure to Fe(III)/AH-. Neither SOD nor catalase protected against AH--stimulated damage. AH--promoted lipid peroxidation was inhibited by butylated hydroxytoluene, a lipophilic antioxidant, but was unaffected by 2,5-dimethylfuran or ethanol, singlet oxygen, and hydroxyl radical traps, respectively. These results suggest that a mechanism exists by which photogenerated LOOHs undergo redox metal-mediated reduction to alkoxy radicals (LO.), which trigger a burst of membrane-disrupting lipid peroxidation.     

Removal of blood group determinants from bovine erythrocyte membranes. 2. Degradation of ghosts by butanol and pyridine

Ghosts produced from erythrocytes collected from six different cattle were degraded with butanol and pyridine. Of a total of 38 different antigenic determinants available for investigation among the six cows, F, V, J and L were the only speci-ficties detected in the subtractions resulting from either method of degradation. After butanol degradation V, J and L antigens were found in the soluble protein fraction, while F was found in the insoluble protein. Pyridine digestion resulted in all four determinants being detected in the sialoprotein layer, while J was found in the lipoprotein as well. All antigens were relatively weak, being detected in inhibition strengths of 10.0 to 1.25 mg/ml.