Interaction of pentoxifylline with human erythrocytes. II. Effects of pentoxifylline on the erythrocyte membrane

The effects of pentoxifylline and other hemorheologically active drugs on the human erythrocyte membrane were examined by means of electron spin resonance spectroscopy. It was observed that the fluidity in the region of the phospholipid head groups in the erythrocyte bilayer was increased by an externally added drug. In this region, membrane fluidity was dependent on the incubation time, suggesting an interaction with membrane proteins. On the other hand, the acyl chain motion in the lower portion of the chain, the hydrophobic end, was reduced in the presence of the drugs. In this case, the acyl chain motion was not time-dependent. These changes of the membrane fluidity at different depths of the membrane induced by the drugs may correlate to the erythrocyte deformability.     

Interaction of pentoxifylline with human erythrocytes. I. Interaction of xanthine derivatives with human erythrocyte ghosts

The interaction of pentoxifylline and other xanthine derivatives with human erythrocyte ghosts was studied. By fluorescence spectroscopy it was found that xanthine derivatives have two modes of binding to erythrocyte ghosts. One is a high-capacity binding to erythrocyte membranes. It seems that the 5-oxohexyl side chain of pentoxifylline is important for this. The second type may be a binding to proteins on the membranes and is specific for pentoxifylline and caffeine. From the circular dichroism spectra, it was presumed that the second binding mode of pentoxifylline occurs at hydrophobic regions of beta-structure of the membrane proteins. The relative high specificity in the interaction of pentoxifylline with erythrocytes should be related to its unique physiological activity on erythrocytes.     

Interaction of liposomes with human erythrocytes

Interaction of liposomes (phospholipid vesicles) with human erythrocytes was studied by means of a spectroscopic method. Transfer of hemoglobin between liposomes and erythrocytes was observed. This transfer was mediated by a migration of band 3 proteins. In this case, a transfer of band 4.5 also was observed by means of electrophoresis. An interaction of lipid monomers from the liposomes with the erythrocyte membranes seemed to be closely correlated to the transfer of these proteins. It was presumed that this interaction induced some changes in the molecular organization of the cell membranes around band 3, resulting in release of the proteins from the erythrocyte membranes.     

Interaction of NAD-dependent dehydrogenases with human erythrocyte membranes

Interaction of D-glyceraldehyde-3-phosphate dehydrogenase (GPDH) and ladate dehydrogenase with human erythrocyte membranes was studied. Under the conditions of low ionic strength, both enzymes bound to the membranes with similar affinities (Kd ≈ 1 μM). The binding was accompanied by complete inhibition of GPDH and by a 65–75% inhibition of ladate dehydrogenase (LDH). Increasing the ionic strength to physiologically meaningful values (0.15M) completely abolished the inactivation of both dehydrogenases in the presence of erythrocyte membranes, but did not preclude their binding. These results suggest that different modes of enzyme-membrane interaction can be realized under the conditions of low and high ionic strength. They also indicate that GPDH and LDH are capable of functioning in a membrane-bound state.     

Analysis of human erythrocyte membrane vesicles produced by shearing

Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and inside-out vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg++-ATPase, Na+,K+-ATPase, acetylcholinesterase, glyceraldehyde-phosphate dehydrogenase, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PAS-staining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PAS-2 at the expense of PAS-1. The bottom vesicle class was very much enriched with PAS-1 at the expense of PAS-2, and PAS-3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluid-mosaic structure.     

Lipid peroxidation of the erythrocyte membrane caused by stimulated polymorphonuclear leukocytes in the presence of ferritin

Lipid peroxidation of erythrocyte membrane was caused by phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocytes (PMN) in the presence of ferritin. PMN themselves were not peroxidized. A lag period was observed before the start of the peroxidation reaction. In contrast, ferritin iron was continuously released by PMA-stimulated PMN, suggesting that accumulation of free iron in the reaction system was important for proceeding of the peroxidation reaction. Superoxide dismutase, catalase, hydroxyl radical scavengers and an iron chelator, diethylenetriaminepenta-acetic acid, inhibited the lipid peroxidation, indicating that the lipid peroxidation is initiated by a hydroxyl radical generated from the interaction of H2O2 with ferrous iron released from ferritin.     

Bilirubin-sensitized photoinactivation of enzymes in the isolated membrane of the human erythrocyte.

Abstract— Bilirubin has been found to sensitize the photodynamic inactivation of several enzymes in the isolated membrane (ghost) of the human red cell. When ghosts (pH 8.0, 10°C) + bilirubin (0.1 mM) were irradiated with blue light (350 Wm^-2), the activity of glyceraldehyde 3-phosphate dehydrogenase decayed with t_1/2? 15 min. No effect was observed in the absence of pigment or with incident yellow light. Diazabicyclo-octane (DABCO) sharply reduced the inactivation rate, suggesting that ¹O₂ is involved. Sodium dodecyl sulfate-gel electrophoresis of ghosts containing fully inactivated glyceraldehyde 3-phosphate dehydrogenase revealed no change in the polypeptide band corresponding to the subunit of the enzyme. Solubilized enzyme, which was similarly photosensitive, could be partially protected by nicotinamide adenine dinucleotide or glyceraldehyde 3-phosphate. The integral enzymes Mg²⁺-ATPase, Na⁺, K⁺-ATPase, and acetylcholinesterase were also affected. Under the above conditions and bilirubin = 0.37 mM, these enzymes were photoinactivated in first-order fashion, k? 2, 1.2 and 0.2 h^-1, respectively. The rate of decay of total ATPase was found to vary as the square root of the bilirubin concentration over the range 7–370 μM. At a fixed bilirubin concentration (0.37 mM), this rate was also shown to be directly proportional to light intensity. Inasmuch as the —SH content of bilirubin-containing ghosts diminished during irradiation, oxidation of essential cysteine residues could be responsible for the inactivation of some of the enzymes studied.     

Heterogeneity in the conformation of different protein fractions from the human erythrocyte membrane.

We have isolated 5 families of proteins from human red blood cell membranes and characterized their secondary structure by ultraviolet circular dichroism measurements. The protein families were prepared by selective solubilization from ghosts under nondenaturing conditions. We find that the intact ghost has a mean alpha-helix fraction of 0.37, whereas a low-ionic-strength extract (bands 1, 2, 5, "spectrin") has a substantially higher helix fraction, 0.55. Further extraction of the ghosts with para-chloromercuribenzoate yields bands 2.1, 4.1, 4.2, and 6; their helix content is only 0.17. Finally, the major intrinsic protein, band 3, was solubilized by a non-ionic detergent. Its helix fraction is 0.38.     

Modification of human erythrocyte membrane potentials by the action of the polyoxyethylene detergent Triton X-100

Triton X-100 in sublytic concentrations causes a marked increase in human erythrocyte membrane permeability to cations with small selectivity to K⁺ ions. This membrane permeabilization induces modification of membrane potential: hyperpolarization in low K⁺ media and depolarization in high K⁺ media. Since the specificity of the K⁺-ionophorous action of Triton X-100 is very low in comparison with valinomycin, the membrane potential changes are only in the range of −9 to +5 mV. It is suggested that they are determined by the generation of a diffusion potential across the membrane, although the influence of Triton X-100 on the potentials at cell surfaces cannot be neglected.     

The exchange of erythrocyte membrane phospholipids with rat liver extracts in vitro.

Intact rat or human erythrocytes and their isolated (ghost) membranes were incubated with the high speed supernatant fraction of homogenates derived from 32P-labeled rat livers. Phospholipid molecules were transferred between the red cell membranes and the liver extracts, as reflected by the convergence of their specific radioactivities with time. Whereas ghosts usually approached isotopic equilibrium with the liver supernatant fraction during a few hours of incubation at 37 degrees C, the exchange of phospholipids by intact cells was no more than one-half, even after 18 hr. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin were all exchanged in both intact cells and ghosts, albeit to different extents. (A control experiment, incubating 32P-labeled rat erythrocytes or ghosts with unlabeled rat liver extracts, also demonstrated the exchange of all four major phospholipids.) These data may signify that the phospholipids on the cytoplasmic side of the membrane of intact erythrocytes do not exchange with the phospholipids in exogenous liver extracts. If so, all four major phospholipid classes would appear to be present to some extent at both membrane surfaces. The first inference is in agreement with several other studies on this membrane, while the second inference is not.     

Interaction of water with the regenerated cellulose membrane studied by DSC

One to three endothermal peaks atributted to melting of bulk and interfacial water were observed by DSC in the regenerated cellulose — water system. The profiles of thermal effects depend on water content, time of conditioning, film pretreatment and the conditions applied during the preceding freezing-thawing cycles. The occurrence might be deduced of melting-crystallisation processes. A large amount of non-freezable strongly bounded water was also detected. Although cellulose absorbs water quickly after immersion, the structural changes consisting on ordering of polymer fraction occur during further conditioning due to increase in strength of water binding. Using the membranes in the separation module at 90°C causes weakening of these bonds. Differences between interaction of particular cellulose films with water can be detected during the first, the second and the third heating. This revised version was published online in July 2006 with corrections to the Cover Date.     

Decomposition of ammonia on rhenium III. Interaction of ammonia with rhenium

The adsorption and steady-state decomposition of ammonia on rhenium has been studied. A mechanism for the interaction of ammonia with the Re surface is suggested.

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Effect of UVA radiation on membrane fluidity and radical decay in human fibroblasts as detected by spin labeled stearic acids

The ultraviolet A (UVA) radiation component of sunlight (320-400 nm) has been shown to be a source of oxidative stress to cells via generation of reactive oxygen species. We report here some consequences of the UVA irradiation on cell membranes detected by electron paramagnetic resonance (EPR) spectroscopy. Paramagnetic nitroxide derivatives of stearic acid bearing the monitoring group at different depths in the hydrocarbon chain were incorporated into human fibroblasts membranes to analyze two main characteristics: kinetics of the nitroxide reduction and membrane fluidity. These two characteristics were compared for control and UVA-irradiated (0-250 kJ/m(2)) cells. The term relative redox capacity (RRC) was introduced to characterize and to compare free radical reduction measured by EPR with some well-known viability/clonogenicity tests. Our results showed that UVA-irradiation produces a more rigid membrane structure, especially at higher doses. Furthermore, we found that trends agree in survival measured by neutral red (NR), trypan blue (TB), and clonogenic efficiency compared with RRC values measured by EPR for low and medium exposure doses. Above 100 kJ/m(2), differences between these tests were observed. Antioxidant effect was modeled by alpha-tocopherol-acetate treatment of the cells before UVA irradiation. While NR, TB and clonogenicity tests showed protection at the highest UVA doses (>100 kJ/m(2)), results obtained with EPR measurements, both membrane fluidity and kinetics, or using MTT test did not exhibit this protective effect.     

Harmonic analysis of large systems. III. Comparison with molecular dynamics

Atomic motions in bovine pancreatic trypsin inhibitor (BPTI), derived from molecular dynamics, harmonic analysis, and quasiharmonic analysis, are compared when a single protein model, energy parameters, and environment are employed. Molecular dynamics (MD) was carried out for 2 nanoseconds. An average structure was determined from the last nanosecond of the MD simulation, when no major structural changes were observed. This structure was used for several harmonic analysis calculations as well as for a reference structure for the quasiharmonic analysis, for both full basis and reduced basis sets. In contrast to the harmonic analysis results, the quasiharmonic reduced basis calculation using a spherical harmonics reduced basis provided good agreement with the full basis calculation, suggesting that when anharmonic effects are considered, BPTI can behave as a homogeneous object. An extensive analysis of the normal modes from a diverse set of 201 minimized MD simulation frames was performed. On only the sub-picosecond time scale were energy minima revisited after a transition to another state. This analysis shows that the dynamics average structure is not representative of the simulation frames in terms of energy and vibrational frequencies. For this model of BPTI, 42% of the motion (mean-squared fluctuation) can be attributed to harmonic limit behavior. A spectral analysis of the correlation function of deformation for a particular normal mode or quasiharmonic mode can be used to determine the time scales of motions which correspond to harmonic vibration, large-scale drift, or sharp transitions between local substrates. © 1995 John Wiley & Sons, Inc.     

A chip calorimetry-based method for the real-time investigation of metabolic activity changes in human erythrocytes caused by cell sickling

Journal of Thermal Analysis and Calorimetry - A new calorimetric technique is described which allows the measurement of metabolic heat rates in biological materials which are triggered by changes...