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.     

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.     

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.     

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.     

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.     

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.     

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.     

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   

ESR study of proton transport across phospholipid vesicle membranes

A new method for measuring the rates of proton transfer through bilayer phospholipid membranes using pH-sensitive nitroxyl radicals is suggested. The pH-sensitive alkylating radical was covalently bound to glutathione. This modified glutathione is pH sensitive at pH 1.5-4.5 and does not penetrate across phospholipid membranes. Using ESR this probe was applied to register the kinetics of pH variations inside large unilamellar phospholipid vesicles after creation of a transmembrane proton gradient. In the acidic region (pH approximately 3) the main mechanism of transmembrane proton transfer is that via transport of a proton in the form of an undissociated acid. The membrane permeability coefficients have been determined for a series of acids (HCl, HClO4, HNO3, upper estimate for H2SO4). Taking into account that imidazoline and imidazolidine nitroxyl radicals can be used as pH probes in a wide range of pH, the present method can be developed for measuring the rates of transmembrane proton transfer in neutral and alkaline media.     

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.