Proteolytic cleavage of band 3 protein in relation to anion transport in fish (Oncorhynchus mykiss) red blood cells

• 1.1. The effects of trypsin and chymotrypsin on HCO₃⁻/Cl⁻ exchange through red blood cell membranes of humans and trout were studied.
• 2.2. To measure the anion exchange we used a right-angle light-scattering technique by applying the Jacobs-Stewart cycle in ammonium solution and the osmotiration method at constant cell volume.
• 3.3. The Cl⁻ flux in human red blood cells remained unaltered after treatment with external trypsin and chymotrypsin while in trout red blood cells the flux decreased.
• 4.4. This partial inhibition of anion transport in fish, ranging from 30 to 40%,suggest that one or several of the cleavage sites in band 3 protein, essential for anion transport function, are exposed in fish red blood cells.
• 5.5. In human red blood cells the fragments of band 3 which are affected by proteolytic digestion, retain their tertiary structure because there is no influence on anion transport.

     

Water diffusion permeability of human erythrocytes studied by a pulsed gradient NMR technique

Water diffusion permeability of human erythrocytes has been measured by NMR using a pulsed magnetic field gradient technique. The measurement of exchange rates was based on restricted diffusion of water molecules within red blood cells. This method avoids addition of paramagnetic ions, such as Mn²⁺ and is used in vivo.The mean lifetime of water inside human erythrocytes was found to be 17 ms at 24°C. A sulfhydryl reagent, known to inhibit water osmotic permeability, reduced significantly water diffusion across the red cell membrane.     

The temperature dependence of erythrocyte water diffusion permeability

1. The activation energy of the diffusion water exchange in red blood cells increases with temperature. 2. Fetal blood has a higher activation energy for diffusion water exchange than adult blood. 3. Treatment of red cells with p-chloromercuribenzoate alters the activation energy and apparently allows a lipid and a protein pore pathway to be resolved. The permeability and activation energy of the treated cells is in the range found for lipid membranes; and the difference between treated and untreated cells, the "protein" pathway, has a diffusion activation energy comparable with that of free water. 4. A resolution of the discrepancies between the NMR methods of measuring diffusion water exchange is suggested.     

The concentration dependence of the hemoglobin mutual diffusion coefficient

Laser correlation Spectroscopy was used to measure the mutual diffusion coefficient, D, of human cyanomethemoglobin (Fe⁺⁺⁺:CN) at varying protein concentrations. These measurements were male at 20°C in a 0.1 M phosphate buffer solution at pH 7.0. For low protein concentrations we find D = (6.43 ± 0.26) × 10^?7 cm²/S and that there is a near linear decrease from this value at higher concentrations. The linear relation between the diffusion coefficient and protein concentration allows us to deduce the value of the linear frictional volume fraction coefficient, K_f= 7.75. and to extrapolate to hemoglobin concentrations equivalent to that in the red blood cell where we estimate D = 4.25 × 10^?7 cm²/s Various theoretical predictions of the dependence of the mutual diffusion coefficient on concentration are tested; we find that the generalized Stokes-Einstein relation can be made to fit our high concentration data if we assume a hard-sphere model and if we include a term involving a hydrodynamic interaction integral.     

Simultaneous determination of superoxide dismutase and catalase in biological materials by polarography.

The polarographic method of catalytic currents applied to a wave of oxygen permits the simultaneous assay of superoxide dismutase and catalase in biological materials with high speed and reproducibility and minimal manipulation of tissues. Washed red blood cells and tissue homogenates give rise to a strong polarographic maximum, apparently due to heme proteins, which interferes with the measurement. This maximum is suppressed by addition of approximately 0.2% plasma. Therefore, the determination of the two enzymes in red blood cells can be carried out by direct addition of whole blood to the polarographic solution. Thirty microliters of blood are enough for optimal determination of both enzymes. The method can determine superoxide dismutase and catalase at concentrations as low as 2 × 10⁻¹¹m and 5 × 10⁻¹⁰m, respectively, and shows a linear correlation between measured activity and enzyme levels. The average values of the two enzymes in human red blood cells was found by this method to be 2.6 × 10⁻⁶m for catalase and 1.8 × 10⁻⁶m for superoxide dismutase, which agree with previously reported values.     

Kinetic characteristics of bicarbonate-chloride exchange across the neonatal human red cell membrane

The kinetics of HCO₃^?/Cl^? exchange across red cell membrane of newborn infants was studied using a stopped-flow rapid reaction apparatus with a glass pH electrode attached. The measured apparent permeability P is (1.35±0.08 (S.E.)) · 10^?4 cm/s (n=30) for newborns, compared with (3.1 ± 0.4) · 10^?4 cm/s (n=15) for adults. These correspond to half-times of 0.2 s for newborns and 0.1 s for adults indicating that neonatal red cells exchange Cl^? for HCO₃^? only half as fast as do adult cells. The temperature dependence of the exchange rate was studied from 2 to 42°C. From the Arrhenius plot the activation energy of the exchange process in neonatal red cells changes from 22.9 kcal/mol (low temperature) to 4.8 kcal/mol (physiological temperature) at a transition temperature of 17°C. These values are lower than the corresponding values for adult red cells, 34.7 and 10.2 kcal/mol. HCO₃^?/Cl^? exchanges in both adult and neonatal red cells are inhibited by phlorizin. Inhibition constants K_i are 0.8 mM and 2.5 mM for adults and newborns, respectively. The differences in the values of the HCO₃^?/Cl^? exchange rate constant and the activation energy of the exchange process between neonatal and adult red cells indicate that there is a modification of HCO₃^?/Cl^? transport system in the neonatal red cell membranes.     

Red Cell Membrane Permeability Deduced from Bulk Diffusion Coefficients

The permeability coefficients of dog red cell membrane to tritiated water and to a series of[¹⁴C]amides have been deduced from bulk diffusion measurements through a "tissue" composed of packed red cells. Red cells were packed by centrifugation inside polyethylene tubing. The red cell column was pulsed at one end with radiolabeled solute and diffusion was allowed to proceed for several hours. The distribution of radioactivity along the red cell column was measured by sequential slicing and counting, and the diffusion coefficient was determined by a simple plotting technique, assuming a one-dimensional diffusional model. In order to derive the red cell membrane permeability coefficient from the bulk diffusion coefficient, the red cells were assumed to be packed in a regular manner approximating closely spaced parallelopipeds. The local steady-state diffusional flux was idealized as a one-dimensional intracellular pathway in parallel with a one-dimensional extracellular pathway with solute exchange occurring within the series pathway and between the pathways. The diffusion coefficients in the intracellular and extracellular pathways were estimated from bulk diffusion measurements through concentrated hemoglobin solutions and plasma, respectively; while the volume of the extracellular pathway was determined using radiolabeled sucrose. The membrane permeability coefficients were in satisfactory agreement with the data of Sha'afi, R. I., C. M. Gary-Bobo, and A. K. Solomon (1971. J. Gen. Physiol. 58:238) obtained by a rapid-reaction technique. The method is simple and particularly well suited for rapidly permeating solutes.     

Transfer of sodium ions across the erythrocyte membrane in manic — Depressive illness: Treatment with lithium carbonate

The efflux of ²²Na from erythrocytes was measured $\text{in vitro}$ under experimental conditions such that rate constants due to efflux either by active transport, passive diffusion, or exchange diffusion could be calculated. No significant differences were found in the rate constants for ²²Na efflux between seven male bipolar depressed patients and eight male control subjects, who had no personal or family history of psychiatric illness. Treatment of patients with lithium carbonate either for less than one week or for 4–5 weeks produced no changes in the rate constants describing the efflux of ²²Na from red cells. Also, addition of 1mM LiCl $\text{in vitro}$ did not alter the active transport of ²²Na from erythrocytes. These data provide no evidence for either an abnormality in ²²Na transfer across the red cell membrane of bipolar depressives or an effect of the lithium ion upon such transfer.     

The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange

The subcellular distribution and kinetic properties of carbonic anhydrase were examined in red blood cells and gills of the lamprey, Petromyzon marinus, a primitive agnathan, and rainbow trout, Oncorhynchus mykiss, a modern teleost, in relation to the evolution of rapid Cl^–/HCO ₃ ^– exchange in the membrane of red blood cells. In the lamprey, which either lacks or has minimal red cell Cl^–/HCO ₃ ^– exchange, there has been no compensatory incorporation of carbonic anhydrase into the membrane fraction of either the red cell or the gill. Carbonic anhydrase activity in red cells is exclusively cytoplasmic, and the single isozyme displays kinetic properties typical of the type I, slow turnover, isozyme. In the red blood cells of the trout, however, which possess high amounts of the band-3 Cl^–/HCO ₃ ^– exchange protein, the single carbonic anhydrase isozyme appears to be kinetically similar to the type II, fast turnover, isozyme. It thus appears that the type I isozyme present in the red blood cells of primitive aquatic vertebrates was replaced in modern teleosts by the kinetically more efficient type II isozyme only after the incorporation and expression of a significant amount of the band-3 exchange protein in the membrane of the red cell.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CA carbonic anhydrase - DTT dithiothreitol - EDTA ethylenediaminetetra-acetate - E ₀ total concentration of free enzyme - i fractional inhibition of enzyme activity - IU international units - K ₁ inhibition constant - K _M Michaelis constant - NBT nitro blue tetrazolium - NCP nitrocellulose paper - RBC red blood cell - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max maximal velocity of reaction     

Deviations from the flux ratio equation for chloride ions in ouabain- and acetazolamide-treated frog skin

In order to establish whether or not chloride ions behave as freely moving particles in “passive”, i.e. ouabain-and acetazolamide-treated, frog skin, tracer fluxes of ³⁶Cl⁻ have been measured while a voltage (generally +40 mV, serosal side positive) across the skin was applied. Ussing's flux ratio equation has been used as a criterion for this type of transport. One group of skin samples exhibited significant exchange diffusion phenomena. Most samples in a second group either behaved according to the flux ratio equation or showed significant and extreme exchange diffusion. From flux ratios obtained at two different voltages across various skin samples, showing extreme exchange diffusion, it appeared that the simple form of Kedem and Essig's law derived from irreversible thermodynamics, which is valid for homogeneous systems, does not apply to the type of exchange diffusion found. The system can, however, be described by a 1 : 1 exchange mechanism working in parallel with a diffusional pathway. The ratio exchange flux/observed efflux must then have a constant value (0.83) at the voltages applied, which implies that the exchange flux is voltage dependent. By comparison with iodide flux experiments as carried out by Ussing, it is shown that iodide exhibits the same type of exchange diffusion. A carrier, possibly responsibe for the observed behaviour, is described.     

Red blood cell dielectrophoresis in axisymmetric fields

Dielectrophoretic velocities of human red blood cells in an axisymmetric field were measured as a function of the applied voltage and the distance from the axis of symmetry. The voltage of the alternating electric field (frequency 2 MHz), applied between two concentric cylindrical metal electrodes (outer and inner radii 0.24 and 1 mm, respectively), was varied up to 19 V. Two kinds of mediums were used: (a) 90% of 2.1% glycine solution and 10% of 5.5% glucose solution and (b) 5.4% sorbitol solution. The results have shown that in both mediums the cell velocities are proportional to the square of the applied voltage and inversely proportional to the cube of the distance from the axis of symmetry, as predicted by the theory. The coefficient of proportionality (dielectrophoretic coefficient) is on the order of 10⁻²⁵ A²s⁴kg⁻¹. It depends on the donor of red blood cells and might be used for diagnostic purposes. These results will be used in future investigations of membrane adhesion, stability and fusion.     

Changes in alanine and glutamine transport during rat red blood cell maturation

Alanine and glutamine transport have been studied during red blood cell maturation in the rat. Kinetic parameters of Na⁺-dependent L-alanine transport were:K _m 0.43 and 1.88 mM andV _max 158 and 45 nmoles/ml ICW/min for reticulocytes and erythrocytes, respectively. During red cell maturation in the rat there is a loss of capacity and affinity of the system ASC for L-alanine transport. The values for Na⁺-dependent L-glutamine transport in reticulocytes wereK _m 0.51 mM andV _max 157 nmoles/ml ICW/min. On the other hand, a total loss of L-glutamine transport mediated by both N and ASC systems is demonstrated in mature red cells. This seems to indicate that during rat red cell maturation the system N disappears. Furthermore, the system ASC specificity in mature cells changes, and glutamine enters the red cell by non-mediated diffusion processes.     

Changes in Amino Acid Transport During Red Cell Maturation

We studied amino acid transport in sheep red blood cells (RBCs) as a function of cell maturation. Transport of amino acids is decreased strikingly in the mature mammalian RBC compared to the immature reticulocyte. Blood obtained 5-6 days after massive bleeding was fractionated on dextran gradients. In the mature erythrocyte amino acids are taken up only slowly, and in the normal experimental interval (60 min) the concentration in the cell does not reach that of the medium. In contrast, the reticulocyte-rich (top) fraction (50-90% reticulocytes) accumulates certain amino acids, particularly histidine, methionine, and leucine. The underlying process is ATP-independent and Na⁺-insensitive, and has properties consistent with exchange diffusion, i.e., accelerated uptake or efflux when unlabeled solute is present on the trans side. The process is apparent not only in intact cells but also in resealed ghosts. The decrease in activity of amino acid transport is a function of red cell maturation. Thus it can be shown that (a) separation of cells according to their density 1, 2, and 3 weeks after bleeding leads to progressively lower amino acid transport activity with increasing cell density; and (b) during in vitro long-term incubation at 37°C of reticulocyte-rich, unfractionated blood (5–10% reticulocytes), amino acid transport decreases while red cell integrity is maintained, as evidenced by the retention of a normal K⁺ gradient and the absence of hemolysis. The progressive loss is seen with resealed ghosts as well as with intact cells. Not all the amino acids examined participate in this exchange process. The most actively exchanged are histidine, leucine, methionine, and phenylalanine. Glycine, proline, arginine, and a-amino isobutyric acid do not participate in the exchange process.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO₂ production after adding H¹⁴CO ₃ ^- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4 diisothiocyanatostilbene-2,2 disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO₂ production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and >200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.     

The basal permeability to water of human red blood cells evaluated by a nuclear magnetic resonance technique

The characteristics of water diffusional permeability (P) of human red blood cells were studied on isolated erythrocytes by a doping nuclear magnetic resonance technique. In order to estimate the basal permeability the maximal inhibition of water diffusion was induced by exposure of red blood cells to p-chloromercuribenzene sulfonate (PCMBS) under various conditions (concentration, duration, temperature). The lowest values of P were around 0.7×10^–3 cm s^–1 at 10°C, 1.2×10^–3 cm s^–1 at 15°C, 1.4×10^–3 cm s^–1 at 20°C, 1.8×10^–3 cm s^–1 at 25°C, 2.1×10^–3 cm s^–1 at 30°C and 3.5×10^–3 cm s^–1 at 37°C. The mean value of the activation energy of water diffusion (E_a,d) was 25 kJ/mol for control and 43.7 kJ/mol for PCMBS-inhibited erythrocytes. The values of P and E_a,d obtained after induction of maximal inhibition of water diffusion by PCMBS can be taken as references for the basal permeability to water of the human red blood cell membrane.     

The restriction of diffusion of cations at the external surface of cardiac myocytes varies between species

In cardiac muscle sarcolemmal structures such as T-tubules, caveolae and negatively charged protein-polysaccharides may affect the rate of cation exchange on the external surface of the cells. To test this hypothesis, we examined the rate of external cation exchange in adult rabbit and rat ventricular myocytes using a rapid solution switcher to change the bulk external solution within 4 ms. To assess the rate of diffusion of monovalent cations, we increased [K⁺]_o from 4.4 to 6.6 or 8.8 mM and measured the time required to achieve a stable membrane depolarization. In rat myocytes, the mean time to 90% depolarization (t₉₀) was significantly longer than that in rabbit myocytes (137 and 64 ms, respectively) and the difference in t₉₀ was not associated with the cell size. To assess the time course of exchange of external Ca²⁺, we rapidly exposed the myocytes to 0 Ca²⁺-2 mM EGTA solution at specific time points before action potentials or voltage clamp steps, and measured the rate of alteration of the normalized peak [Ca²⁺]_i transient (Flux-3) or Ca²⁺ current. Exposure to 0 Ca²⁺-2 mM EGTA solution caused a decline in the intracellular calcium transient. In rat myocytes, the rate of decline in the [Ca²⁺]_i transient was much slower (t₉₀ > 1500 ms, the time required for 90% decline) than for the rabbit (t₉₀ = 295 ms). Also, the rate of decline in the Ca²⁺ current was prolonged in rat myocytes (t₉₀ = 910 ms) compared with rabbit myocytes (t₉₀ = 241 ms). These data indicate that there is a restricted space on the external surface of sarcolemma which limits diffusion of divalent cations more markedly than monovalent cations. The extent of this limitation of cation diffusion varies between species, and may have functional significance.     

Murine antigen H-2.7: In vitro phenotypic conversion of erythrocytes

The H-2.7 antigen in normal mouse serum can be passively adsorbed to H-2.7^– erythrocytes in 10 percent sucrose (low ionic strength) solution. This antigen can also be stripped off the H-2.7⁺ erythrocytes under the same conditions provided the H-2.7⁺normal serum is absent. The stripped red blood cells can regain the H-2.7 antigen upon reincubation with H-2.7⁺ normal serum. The attachment of the H-2.7 antigen to erythrocytes probably occurs via a specific receptor.Abbreviations used in this paper BSA bovine serum albumin - B10 C57BL/10Sn - HA hemagglutination - LIS low ionic strength solution - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

Murine antigen H-2.7: Phenotypic conversion of erythrocytes in radiation chimeras

By use of radiation chimeras produced between H-2.7⁺ and H-2.7^}- strains, A.SW and A.BY and B10.S(7R) and B10.S(9R), we demonstrate that the H-2.7 antigen can be passively attached to or detached from red blood cells. Thus, genetically H-2.7^}- red blood cells derived from H-2.7^}- bone marrow cells, gain H-2.7 antigen while maturing in the H-2.7⁺ host. Similarly, genetically H- 2.7⁺ red blood cells derived from H-2.7⁺ bone marrow cells become H-2.7^}- while maturing in H-2.7^– recipients. This behavior of the H-2.7 antigen is similar to that described for human Chido and Rodgers blood group antigens.Abbreviations used in this paper BMT bone marrow transfer - BSA bovine serum albumin - CT cytotoxicity test - HA hemagglutination - HBSS10 Hank's balanced salt solution containing 10% fetal calf serum - NMS normal mouse serum - PBS phosphate-buffered saline - PVP polyvinylpyrrolidone - RBCs red blood cells