Study of amino and sulfhydryl sites in the sodium pathway in dog red blood cell membranes

Summary Amino reactive TNBS (2,4,6-trinitrobenzene sulfonic acid), SITS (4-acetamido-4-isothiocyano-stilbene-2-2-disulfonic acid), and Zn⁺⁺, and SH reactive Hg⁺⁺ were employed to study sodium channels in dog red blood cells. Simultaneous modification of the membrane with both a SH and an amino modifier results in an increase in Na⁺ permeability which is equal to the sum of their individual effects. This indicates that SH and amino sites are separate units. Three lines of evidence indicate that the amino sites are more superficial than the SH sites. (1) Pretreatment with an amino modifier decreases the effectiveness of subsequent SH modification. (2) SITS, a nonpenetrating amino reagent, enhances Na⁺ permeability while DTNB, a nonpenetrating SH modifier, is ineffective. (3) Pretreatment of amino sites decreases the apparent affinity of Hg⁺⁺ for SH sites. In addition, three lines of evidence indicate that TNBS and Zn⁺⁺ modify different amino sites. First, simultaneous modification with TNBS and Zn⁺⁺ results in an increase in Na⁺ permeability equal to the sum of their individual effects. Secondly, Zn⁺⁺ causes an increase in Na⁺ permeability in cells previously treated with TNBS. Finally, the pH dependence of Zn⁺⁺ modification is opposite that for TNBS modification. These pH experiments suggest that Zn⁺⁺ enhances Na⁺ permeability by reacting with unprotonated amino sites while TNBS modifies protonated amino sites. It is concluded that the sodium permeability of dog red blood cells is normally limited by superficial amino sites and deeper sulfhydryl sites in the sodium channels.     

Sodium and calcium movements in dog red blood cells

Determinants of 45Ca influx, 45Ca efflux, and 22Na efflux were examined in dog red blood cells. 45Ca influx is strongly influenced by the Na concentration on either side of the membrane, being stimulated by intracellular Na and inhibited by extracellular Na. A saturation curve is obtained when Ca influx is plotted as a function of medium Ca concentration. The maximum Ca influx is a function of pH (increasing with greater alkalinity) and cell volume (increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca is stimulated by increasing concentrations of extracellular Na. 22Na efflux is stimulated by either Ca or Na in the medium, and the effects of the two ions are mutually exclusive rather than additive. Quinidine inhibits Ca-activated 22Na efflux. The results are considered in terms of a model for Ca-Na exchange, and it is concluded that the system shows many features of such a coupled ion transport system. However, the stoichiometric ratio between Ca influx and Ca-dependent Na efflux is highly variable under different experimental conditions. Because the Ca fluxes may reflect a combination of ATP-dependent, outward transport and Na-linked passive movements, the true stoichiometry of an exchanger may not be ascertainable in the absence of a specific Ca pump inhibitor. The meaning of these observations for Ca-dependent volume regulation by dog red blood cells is discussed.     

Enhancement of anion permeability in lecithin vesicles by hydrophobic proteins extracted from red blood cell membranes.

Triton X-100 extracts of membrane proteins from ghosts of normal and pronase treated cells enhance the anion permeability of lecithin vesicles. With proteins from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate), a specific inhibitor of anion transport, the anion permeability is not enhanced. On the basis that the Triton X-100 extracts are considerably enriched in a protein component of 95,000 molecular weight (or a 65,000 molecular weight segment in the case of pronase treated cells), and that DIDS is bound almost exclusively to the same proteins, it is suggested that the pronase resistant, 65,000 molecular weight segment of the 95,000 molecular weight protein is directly involved in anion transport.     

The permeability of human red blood cell membranes to hydrogen peroxide is independent of aquaporins

Hydrogen peroxide (H₂O₂) not only is an oxidant but also is an important signaling molecule in vascular biology, mediating several physiological functions. Red blood cells (RBCs) have been proposed to be the primary sink of H₂O₂ in the vasculature because they are the main cellular component of blood with a robust antioxidant defense and a high membrane permeability. However, the exact permeability of human RBC to H₂O₂ is neither known nor is it known if the mechanism of permeation involves the lipid fraction or protein channels. To gain insight into the permeability process, we measured the partition constant of H₂O₂ between water and octanol or hexadecane using a novel double-partition method. Our results indicated that there is a large thermodynamic barrier to H₂O₂ permeation. The permeability coefficient of H₂O₂ through phospholipid membranes containing cholesterol with saturated or unsaturated acyl chains was determined to be 4 × 10⁻⁴ and 5 × 10⁻³ cm s⁻¹, respectively, at 37 °C. The permeability coefficient of human RBC membranes to H₂O₂ at 37 °C, on the other hand, was 1.6 × 10⁻³ cm s⁻¹. Different aquaporin-1 and aquaporin-3 inhibitors proved to have no effect on the permeation of H₂O₂. Moreover, human RBCs devoid of either aquaporin-1 or aquaporin-3 were equally permeable to H₂O₂ as normal human RBCs. Therefore, these results indicate that H₂O₂ does not diffuse into RBCs through aquaporins but rather through the lipid fraction or a still unidentified membrane protein.     

Reflection coefficients of permeant nonelectrolytes for dog and beef red cell membranes.

The reflection coefficient, sigma, for several small permeant nonelectrolytes was determined for dog and beef red blood cell membranes. Our sigma values were considerably higher than those previously reported for dog cells; e.g., out sigma urea was 87% higher than the sigma urea of Rich, Sha'afi, Barton and Solomon (J. Gen. Physiol. 50: 2391, 1967). Our sigma values for urea were only slightly greater in beef cells than previously reported by Farmer and Macey (Biochim. Biophys. Acta 290: 290, 1972). We found that a trend exists when (1 - sigma) is plotted against the log of the permeability coefficient, omega. This observation is also consistent with our previously reported sigma data for human red cell membranes (Owen & Eyring, J. Gen. Physiol. 66: 241, 1972). This trend suggests that small hydrophilic molecules interact highly with cell membrane water. The exceptions to this trend were lipophilic molecules, indicating they do not interact with water while penetrating the red cell membrane.     

On the structure of agglutinated sheep red blood cell membranes.

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160-186 A. It is concluded that two asymmetric membranes are contained in the elementary period. Lipid phases with preferentially oriented hydrocarbon chains are part of the membrane structure. The stacking of the membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 A-1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.     

Sodium movements in the human red blood cell

Measurements were made of the sodium outflux rate constant, ^o k _Na, and sodium influx rate constant, ⁱ k _Na, at varying concentrations of extracellular (Na_o) and intracellular (Na_c) sodium. ^o k _Na increases with increasing [Na_o] in the presence of extracellular potassium (K_o) and in solutions containing ouabain. In K-free solutions which do not contain ouabain, ^o k _Na falls as [Na_o] rises from 0 to 6 mM; above 6 mM, ^o k _Na increases with increasing [Na_o]. Part of the Na outflux which occurs in solutions free of Na and K disappears when the cells are starved or when the measurements are made in solutions containing ouabain. As [Na_o] increases from 0 to 6 mM, ⁱ k _Na decreases, suggesting that sites involved in the sodium influx are becoming saturated. As [Na_c] increases, ^o k _Na at first increases and then decreases; this relation between ^o k _Na and [Na_c] is found when the measurements are made in high Na, high K solutions; high Na, K-free solutions; and in (Na + K)-free solutions. The relation may be the consequence of the requirement that more than one Na ion must react with the transport mechanism at the inner surface of the membrane before transport occurs. Further evidence has been obtained that the ouabain-inhibited Na outflux and Na influx in K-free solutions represent an exchange of Na_c for Na_o via the Na-K pump mechanism.     

Melittin-induced alterations in dynamic properties of human red blood cell membranes.

The interaction of bee venom melittin with erythrocyte membrane ghosts has been investigated by means of fluorescence quenching of membrane tryptophan residues, fluorescence polarization and ESR spectroscopy. It has been revealed that melittin induces the disorders in lipid-protein matrix both in the hydrophobic core of bilayer and at the polar/non-polar interface of melittin complexed with erythrocyte membranes. The peptide has been found to act most efficiently at the concentration of the order of 10(-10) mol/mg membrane protein. The apparent distance separating the membrane tryptophan and bound 1-anilino-8-naphthalenesulphonate (ANS) molecules is decreased upon melittin binding, which results in a significant increase of the maximum energy transfer efficiency. Significant changes in the fluorescence anisotropy of both 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalenesulphonate bound to erythrocyte ghosts, which have been observed in the presence of melittin and crude venom, indicate membrane lipid bilayer rigidization. The effect of crude honey bee venom has been found to be of similar magnitude as the effect of pure melittin at the concentration of 10(-10) mol/mg membrane protein. Using two lipophilic spin labels, methyl 5-doxylpalmitate and 16-doxylstearic acid, we found that melittin at its increasing concentrations induces a well marked rigidization in the deeper regions of lipid bilayer, whereas the effect of rigidization near the membrane surface maximizes at the melittin concentration of 10(-10) mol/mg (10(-4) mol melittin per mole of membrane phospholipid). The decrease in the ratio hw/hs of maleimide and the rise in relative rotational correlation time (tau c) of iodacetamid spin label, indicate that melittin effectively immobilizes membrane proteins in the plane of the lipid bilayer. We conclude that melittin-induced rigidization of the lipid bilayer may induce a reorganization of lipid assemblies as well as the rearrangements in membrane protein pattern and consequently the alterations in lipid-protein interactions. Thus, the interaction of melittin with erythrocyte membranes is supposed to produce local conformational changes in membranes, which are discussed in the connection with their significance during the synergistic action of melittin and phospholipase of bee venom on red blood cells.     

The passive permeability of the red blood cell in cations

The efflux of salt from human red blood cells suspended in isotonic sucrose plus low concentrations of salt, was measured under steady-state conditions. The relationship between the efflux and the log of the salt concentration can be fitted by two straight lines with a sharp inflection point, the steeper slope occurring at concentrations below 0.2 mM NaCl. The determining factor in the rate of efflux is the ionic strength rather than the specific monovalent cations or anions and the effects are completely reversible. With an increase in temperature, the effects of reduced ionic strength are more pronounced and the inflection point is shifted toward higher salt concentrations. An increase in pH leads to an increased efflux at a given ionic strength, but the size of the pH effect is small at low ionic strength. At a given pH, the data can be fitted by a simplified form of the Goldman equation suggesting that with reduction in ionic strength, the permeability remains constant until the inflection point is reached. At that ionic strength, a sharp reversible transition to a new permeability state occurs. The permeability increases with an increase in the external but not the internal pH.     

Pyruvate kinase-catalyzed ATP-formation in human red blood cell membranes

Previous studies on the linkage between enzymatically catalyzed ATP-generating reactions in the red blood cell membrane and the sodium and potassium transport in the control of overall glycolysis of human erythrocytes were controversial. In this study a significant amount of pyruvate kinase activity is shown to be localized within the membrane. Membrane fragments produce 20.5 mumol of ATP per 10(10) membranes per hour from phosphoenolpyruvate and ADP. The kinetics of the membrane-localized pyruvate kinase do not differ from those of the enzyme from hemolysates. The results clearly document the presence of the second ATP-generating enzyme of glycolysis, pyruvate kinase, in human red blood cell membranes. The main fraction of the enzyme is deeply hidden in the lipid layers of the membrane. It can be demasked by mechanical desintegration of membranes at high levels of activity. It is suggested that the amount of the membrane-localized fraction of pyruvate kinase is related to the clinical severity of the hemolytic process in pyruvate kinase deficiency.     

Conductometric study of shear-dependent processes in red cell suspensions. I. Effect of red blood cell aggregate morphology on blood conductance

The conductance and capacitance of flowing and quiescent red blood cell (RBC) suspensions were measured at a frequency of 0.2 MHz. The results demonstrate that the time-dependent changes in the conductance recorded during the aggregation process differ in nature for suspensions of short linear rouleaux, branched aggregates and RBC networks. It is shown that the conductance of RBC suspensions measured during the aggregation and disaggregation processes follows the morphological transformations of the RBC aggregates. Thus, this method enables characterization of the morphology of RBC aggregates formed in whole blood and in suspensions with physiological hematocrits both under flow conditions and in stasis. These results in combination with previous ones suggest that this technique can be used for studies of dynamic RBC aggregation and probably for diagnostic use.     

On the structure of agglutinated sheep red blood cell membranes

Specimens of isolated sheep red blood cell membranes are prepared by an agglutination technique in which membranes are stacked in regular arrays. X-ray diffraction patterns are recorded from such specimens which show meridional and equatorial diffraction phenomena. The meridional reflections correspond to single lamellar repeat periods of 160–186 Å. It is concluded that two asymmetric membranes are contained inthe elementary period. Lipid phases with preferentialyl oriented hydrocarbon chains are part of the membrane structure. The stacking of membranes is also demonstrated in the electron microscope. The X-ray scattering curve of intracellular hemoglobin of intact sheep red blood cells is recorded to a spacing of about 8 Å^?1. The broad diffraction rings of this scattering curve are replaced by a series of rather sharp rings, when the red blood cells are agglutinated and placed in a hypertonic medium. Both the presence of a functioning membrane and the agglutination appear to be essential for the full expression of this phenomenon.