Se-mediated domain-domain communication in Band 3 of human erythrocytes

Na₂SeO₃ could affect the anion flux of Band 3 of inside-out erythrocyte membrane vesicles (IOVs). Such effect was believed to be based on the interaction of SH groups of Band 3 with Na₂SeO₃. This effect could be eliminated when the cytoplasmic domain of Band 3 was proteolytically removed by trypsin. This suggested that SH groups in the cytoplasmic domain were involved in such interaction. Measurement of the pH dependence of intrinsic fluorescence intensity provided evidence that conformational changes of Band 3 occurred as a consequence of interaction with selenite. KI quenching of intrinsic fluorescence of Band 3 could also show that there was a conformational change in the cytoplasmic domain of Band 3 after reaction with Na₂SeO₃. Such conformational change in turn could be transmitted to the membrane domain of Band 3 monitored by quenching of intrinsic fluorescence of Band 3 using hypocrellin B (HB) (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China). It is suggested that the cytoplasmic domain of Band 3 is not necessary for its anion flux, but is essential for the regulation (e.g., by Se) of its active site located at the membrane domain, and hence, it may provide evidence of communication between the cytoplasmic domain and the membrane domain of Band 3.     

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.     

Effect of glycyrrhetinic acid on membrane band 3 in human erythrocytes

Glycyrrhetinic acid (GA) is a hydrolytic product of the triterpene glycoside of glycyrrhizic acid, one of the main constituents of licorice root, which has long been studied, due to its several biological and endocrine properties. In this paper, GA was tested on human erythrocytes, and GA-induced alterations were compared with those caused by diamide, a mild oxidant inducing well-characterized cell/membrane alterations, and n-ethylmaleimide (NEM), as alkylating agent. In order to verify the biochemical steps underlying the action of GA, band 3 Tyr-phosphorylation level, enzyme recruitment and band 3 clustering in cells pre-incubated with GA before diamide treatment were all examined. Results show that GA, in a dose-dependent manner, prevents both diamide and NEM-induced band 3 Tyr-phosphorylation, but not GSH decrease caused by both compounds. In addition, diamide-induced band 3 clustering and IgG binding to altered cells were also completely reversed by GA pre-treatment. Also, when membrane sensitivity toward proteolytic digestion was tested, GA-treated cells showed high resistance to proteolysis. In conclusion, in human erythrocytes, GA is proposed to strengthen membrane integrity against both oxidative and proteolytic damage.     

Detergent-resistant membranes in human erythrocytes and their connection to the membrane-skeleton

In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to existin vivo in the form of lipid ‘rafts’, microdomains enriched in cholesterol and sphingolipids, and in specific classes of proteins, that appear to play specialized roles for signal transduction, cell-cell recognition, parasite or virus infection, and vesicular trafficking. These structures are operationally defined as membranes resistant to solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). This definition appears to be necessary and sufficient, although additional manoeuvres, not always described with sufficient detail, may be needed to ensure isolation of DRMs, like mechanical homogenization, and changes in the pH and/or ionic strength of the solubilization medium. We show here for the human erythrocyte that the different conditions adopted may lead to the isolation of qualitatively and quantitatively different DRM fractions, thus contributing to the complexity of the notion itself of lipid raft. A significant portion of erythrocyte DRMs enriched in reported lipid raft markers, such as flotillin-1, flotillin-2 and GM₁, is anchored to the spectrin membrane-skeleton via electrostatic interactions that can be disrupted by the simultaneous increase in pH and ionic strength of the solubilization medium. An erratum to this article is available at .     

Simulations of NMR-detected diffusion in suspensions of red cells: the effects of variation in membrane permeability and observation time

Monte Carlo random-walk simulations of diffusion in virtual lattices of cells have been used to study and characterize diffusion-coherence phenomena that arise when pulsed field-gradient spin-echo (PGSE) nuclear magnetic resonance (NMR) experiments are conducted on human red blood cell (RBC; erythrocytes) suspensions. These coherence effects are manifest as diffraction-like patterns when the normalized PGSE signal intensities are plotted as a function of the spatial wave vector q in so-called q-space plots. q-Space analysis is sensitive to small changes in cell morphology, cell size, membrane transport rates, hematocrit, and packing arrangement. In the present study we used simulations to predict the effect of varying the time over which diffusion is measured (the "observation time" or "diffusion time") and the permeability of the membrane on the form of q-space plots. Thus we predict that inhibiting water exchange across the human RBC membrane, such that the value of the permeability coefficient is reduced by approximately an order of magnitude below the normal physiological value, will effectively render the membrane impermeable on the timescale of the PGSE NMR experiment; further inhibition will therefore result in negligible reduction in the measured root-mean-square displacement (r.m.s.d.) of diffusing water as a function of the observation time. The work also underscores the importance of using an appropriate experimental observation time if q-space data are to be used to estimate compartment dimensions and interbarrier spacing, and illustrates an expeditious method for determining this value.     

1H NMR methods for the noninvasive study of metabolism and other processes involving small molecules in intact erythrocytes

1H NMR methods are described with which resolved resonances can be obtained for many of the small molecules in intact erythrocytes. In one method, the more intense hemoglobin resonances are suppressed by transfer of saturation throughout the hemoglobin spin system by cross relaxation following a selective saturation pulse. In a second method, the hemoglobin resonances are eliminated with the spin-echo pulse sequence by using a between-pulse delay time long enough for complete elimination of the hemoglobin resonances by spin-spin relaxation. Selected examples of the study of erythrocyte biochemistry by 1H NMR are discussed.     

Alterations in membrane permeability of malaria-infected human erythrocytes are related to the growth stage of the parasite

During the intraerythrocytic growth of Plasmodium falciparum in culture, marked changes are observed in the permeability properties of the host cell membrane. Anionic substances otherwise impermeant to normal cells, become highly permeant to infected cells. These changes in permeability become apparent as rings mature into trophozoites and remain throughout schizogony. The permeability changes to anionic substances are not manifested as degradation of band 3, the purported erythrocyte anion transporter. They probably reflect alterations of a more general nature.     

Membrane perturbations induced by the interactions of zinc ions with band 3 in human erythrocytes

Of group 12 metals, zinc is an essential element to maintain our life, but other metals such as cadmium and mercury are toxic in cellular activities. Interactions of these metals with biomembranes are important to understand their effects on our living cells. Here, we describe the membrane perturbations induced by these metals in human erythrocytes. Of these metals, Zn²⁺ ions only induced the erythrocyte agglutination. Histidine residues in extracellular domains of band 3 participated in Zn²⁺-induced agglutination. Interestingly, it was found that band 3-cytoskeleton interactions play an important role in Zn²⁺-induced agglutination. In contrast with Hg²⁺ and Cd²⁺ ions, Zn²⁺ ions greatly suppressed pressure-induced hemolysis by cell agglutination. Such a suppression was removed upon dissociation of agglutinated erythrocytes by washing, indicating the reversible interactions of Zn²⁺ ions with erythrocyte membranes. Excimer fluorescence of pyrene indicated that spectrin is denatured by a pressure of 200 MPa irrespective of hemolysis suppression. Taken together, these results suggest that the agglutination of erythrocytes due to the interactions of Zn²⁺ ions with band 3 is stable under pressure, but spectrin, cytoskeletal protein, is denatured by pressure     

Internal motion time scales of a small, highly stable and disulfide-rich protein: A 15N, 13C NMR and molecular dynamics study

Motions of the backbone CH and threonine CH bonds of toxin were investigated using natural abundance 13C NMR and molecular dynamics. Measurement of the 13C longitudinal and transverse relaxation rates employed ACCORDION techniques together with coherence selection by pulsed field gradients and sensitivity enhancement through the use of preservation of equivalent pathway, thus allowing a considerable reduction of the required spectrometer time. 13C R1, R2, 1H13C NOE were obtained, as well as the variations of R1(90° ) as a function of the rf field strength. These data were compared to those recorded by 1H and 15N NMR on a labelled sample of the toxin [Guenneugues et al. (1997) Biochemistry, 36, 16097–16108]. Both sets of data showed that picosecond to nanosecond time scale motions are well correlated to the secondary structure of the protein. This was further reinforced by the analysis of a 1 ns molecular dynamics simulation in water. Several CH and threonine CH experimentally exhibit fast motions with a correlation time longer than 500 ps, that cannot be sampled along the simulation. In addition, the backbone exhibits motions on the microsecond to millisecond time scale on more than half of its length. Thus, toxin , a highly stable protein (Tm=75°C at acidic pH) containing 61 amino acids and 4 disulfides, shows important internal motions on time scales ranging from 0.1–0.5 ps, to 10–100 ps, 1 ns, and about 30 s to 10 ms.     

C NMR relaxation studies of 1:2 LiCl-ethylaluminum dichloride solutions

A new room-temperature molten salt, 1:2 LiCl-ethylaluminum dichloride (LiCl-EtAlCl₂, f.p. about 178 K), is examined using ¹³C relaxation methods at 7.05 T (−25 to + 80 °C). The methylene carbon undergoes scalar relaxation of the ‘second kind’ as it is coupled to a faster relaxing (quadrupolar) nucleus. LiCl-EtAlCl₂ undergoes a significant liquid-state phase change between 5 and 15 °C as evidenced by observed changes in the relaxation properties of the methylene and methyl carbons and J(¹³C−²⁷Al). The J(¹³C−²⁷Al) coupling constants are 75 (− 10 to + 5 °C) and 11 Hz (15–65 °C), indicating a change in structure between 5 and 15 °C. Chemical shift anisotropies of 56 and 48 ppm are obtained for the methylene and methyl carbons in the EtAlCl₂ dimer part of the 1:2 LiCl-EtAlCl₂ solution.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

Using simple 13C NMR linewidth and relaxation measurements to make detailed chemical shift assignments in triacylglycerols and related compounds

Two simple experiments measuring the ¹³C linewidths ν_1/2 and spin–lattice relaxation times T₁ of each of the signals in the spectrum of trilinolein indicate that the ν_1/2 and T₁ values are consistent with the different degrees of motional freedom expected for the various ¹³C nuclei. However, for each chain, the ν_1/2 and T₁ measurements indicate a small reversal in mobility at C-10 relative to C-9 before motional freedom again steadily increases on each chain starting at C-11. The T₁ experiment allows unambiguous assignments of the C-8 signal and C-14 signal, which differ by only 0.010 ppm. Measurements of ¹³C ν_1/2 and T₁ values on tripalmitin provide secure assignments for the C-5 and C-6 signals, for which conflicting assignments have been reported. The T₁ measurements also show that among the tightly clustered C-8 through C-12 signals, the C-11 signals are the most downfield, while the C-12 signals are the most upfield, again contrary to a previous report. Similar measurements of ¹³C ν_1/2 and T₁ values on other triacylglycerols or related compounds may prove equally useful in making chemical shift assignments and detecting any discontinuities in motional freedom along a chain. The benefits and possible limitations of ultrahigh field NMR for studying triacylglycerols and related compounds are discussed.     

Effects of anesthetic alcohols on membrane transport processes in human erythrocytes

1. 1. Anesthetic alcohols (pentanol, hexanol and heptanol) were found to increase the fluidity of red cell membrane lipids as monitored by the fluorescence depolarization of diphenylhexatriene. The relative potency of the alcohols was found to be parallel to their relative membrane/water partition coefficients.

2. 2. Hexanol had biphasic effect on erythritol uptake by simple diffusion by red cells. At concentrations less than 9 mM, hexanol had no significant effect. At concentrations greater than 9 mM, there was an approximately linear increase in erythritol permeability with increasing alcohol concentration.

3. 3. The facilitated transport of uridine was markedly inhibited by hexanol. Hexanol at 6 mM produced a 65% inhibition of uridine (4 mM) uptake. Hexanol decreased both the apparent K_m and V values for the equilibrium exchange of uridine.

4. 4. The facilitated transport of galactose was only slightly inhibited by hexanol.

5. 5. Hexanol was without effect on the passive and active fluxes of Na⁺ and K⁺ in red cells with altered cation contents. Cells that were slightly depleted of K⁺ and cells that were highly K⁺-depleted were both insensitive to hexanol.

Hemolysis of human erythrocytes induced by melamine-cyanurate complex

Melamine is a widely-used chemical in industries. In recent years, melamine has been found to be involved in outbreaks of renal injury in infants and animals. Pathological studies indicated that the melamine-induced acute renal failure was related to the concurrence of melamine and other triazine analogs such as cyanuric acid. In the present study, human erythrocytes were used as an in vitro model to explore the cytotoxicity of melamine and its complex with cyanuric acid. The results demonstrated that mixing melamine and cyanuric acid resulted in the formation of insoluble particles and that the insoluble melamine-cyanurate complex induced membrane damages of human erythrocytes. The membrane damages included hemolysis, K⁺ leakage, alterations in cell shape and membrane fragility, and inhibition of enzymatic activity. By contrast, either melamine or cyanuric acid alone had no effect on erythrocyte membranes. The results of this study may provide a fresh insight into the melamine toxicology.     

Role of phospholipids in the DDT-induced efflux of potassium in human erythrocytes

Incubation of human erythrocytes for 1–2 h at 37°C in a suspension of dipalmitoylphosphatidylcholine (DPPC) liposomes results in a phospholipid enrichment of erythrocyte membranes by 45–55% and a depletion of cholesterol by 19–24%. The enrichment by DPPC was time and concentration dependent. By contrast, dioleoylphosphatidylcholine (DOPC) liposomes were less effective in enriching the membranes with phospholipid and in depleting the membranes of cholesterol. Concomitantly, the DDT-induced efflux of K⁺ was reduced in the case of DPPC-enriched erythrocytes but enhanced in DOPC-enriched erythrocytes. These results suggest that DDT partitions more readily into the unsaturated than the saturated phospholipids of the erythrocyte membrane. It is concluded that the extent to which DDT affects the flux of K⁺ across the membrane is dependent on the fluidity of the lipid phase. We also report here a rapid method for cholesterol depletion of red blood cells in comparison to previously reported methods.     

Characterization of the Band 3 substrate site in human red cell ghosts by NDS-TEMPO,a disulfonatostilbene spin probe: The function of protons in NDS-TEMPO and substrate-anion binding in relation to anion transport

Summary NDS-TEMPO is a specific disulfonatostilbene spin label for the Band 3 substrate site (K. F. Schnell, W. Elbe, J. Käsbauer & E. Kaufmann,Biochim. Biophys. Acta 732:266–275, 1983). The pH dependence of NDS-TEMPO binding and of chloride and sulfate binding was studied in resealed human erythrocyte ghosts. pH was varied from 6.0 to 9.0. The ESR spectra from NDS-TEMPO-labeled red cell ghosts exhibited a strong immobilization of membrane-bound NDS-TEMPO. Changes of pH had no effect upon the mobility of membrane-bound NDS-TEMPO. A mutual competition between NDS-TEMPO binding and the binding of the substrate-anions, chloride and sulfate, was observed throughout the entire pH range. The maximal number of NDS-TEMPO binding sites per cell was in the range of 9.0×10⁵ to 1.10×10⁶ and was found to be insusceptible to changes of pH. The NDS-TEMPO/substrate-site and the chloride/substratesite dissociation constants amounted to 1.25 m and to 17mm and were independent of pH from pH 6.0 to 8.0, while the sulfate/substrate-site dissociation constant displayed a strong pH dependency with a maximum of 50mm at about pH 7.0. The NDS-TEMPO inhibition constants from the chloride and the sulfate flux experiments were 0.5 m (0°C) and 1.8 m (25°C), respectively, and are in close accordance with the NDS-TEMPO/substrate-site dissociation constants. Our studies provide strong evidence for the assumption that NDS-TEMPO binds in fact to the substrate site of Band 3. They show that the strong pH dependence of the chloride and of the sulfate transport cannot result from the pH dependency of substrate-anion binding, but point to the participation of ionizable regulator sites in transport catalysis. These regulator sites seem to be positioned outside the substrate site of the Band 3 transport domain.     

The influence of temperature and incubation time on deformability of human erythrocytes

Human erythrocytes have been heated and stressed in a novel and controlled manner using rectangular microcapillaries. Heated cells attached to the capillary wall were stressed by liquid flow. Under particular conditions of stress, temperature and incubation time the body of the cell could be pulled in the flow, retaining a connection with the glass by means of a narrow process or tether. The tethers appear as: regularly beaded, irregularly beaded or without beads depending upon the incubation conditions. We have outlined the incubation regimes necessary to achieve these different responses in the temperature range 48–55°C. The cells become less deformable as the incubation is continued beyond an optimum time. The behaviour of the tether is compared with that of a viscoelastic liquid. Circular dichroism studies of ghost membranes show that the denaturation of membrane proteins is partially reversible when incubation times are similar to those required to bring about a loss of deformability.     

13C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

The widespread importance of induced fit and order-disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein-RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report (13)C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3'-untranslated region (3'-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure (13)C T(1), T(1rho) and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T(1rho) at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region "breathes" on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.     

An allosteric pore model for sugar transport in human erythrocytes

Glucose transport in human erythrocytes is characterized by a marked asymmetry in the $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for entry and for exit. In addition, they show a high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and a high $\text{V}$ for equilibrium exchange but low $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for infinite cis and for infinite trans exit and entry. An allosteric pore model has been proposed to account for these characteristics. In this model, substrate-induced conformational changes destabilize the interfaces between protein subunits (the pore gates).Pores doubly occupied from inside destabilize the transport gates and result in high $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ and high $\text{V}$ transport parameters. This effect is less marked when pores are doubly occupied from outside and therefore transport asymmetry results.