Role of Surface Electric Charge in Red Blood Cell Interactions

The role of the surface charge of human red blood cells (RBC's) in affecting RBC aggregation by macromolecules was studied by comparing the behavior of normal RBC's with that of RBC's treated with neuraminidase, which removes the sialic acids from the cell membrane and reduces the zeta potential. RBC aggregation in dextrans with different molecular weights (Dx 20, Dx 40, and Dx 80) was quantified by microscopic observation, measurement of erythrocyte sedimentation rate, and determination of low-shear viscosity. Dx 20 did not cause aggregation of normal RBC's, but caused considerable aggregation of neuraminidase-treated RBC's. Neuraminidase-treated RBC's also showed stronger aggregation than normal RBC's in Dx 40 and 80. Together with the electron microscopic findings that the intercellular distance in the RBC rouleaux varies with the molecular size of dextrans used, the present study indicates that the surface charge of RBC's inhibits their aggregation by dextrans and that the electrostatic repulsive force between cell surfaces may operate over a distance of 20 nm.     

A monolayer and freeze-etching study of charged phospholipids. I. Effects of ions and pH on the ionic properties of phosphatidylglycerol and lysylphosphatidylglycerol

A comparative study of the ionic properties of phosphatidylglycerol (PG) and lysylphosphatidylglycerol (LPG) has been carried out using monolayer and freeze-etch techniques. It is shown that the ionization state of the PG monolayer is strongly dependent on the subphase ionic strength. Mg⁺⁺ and Ca⁺⁺ induced a marked condensing effect. With Ca⁺⁺ a typical cylindrical structure could be observed by freeze etching, this structure being assumed to be generated by Ca⁺⁺ binding to PG. These phenomena were observed with both didodecanoyl-PG and PG from S. aureus. With respect to LPG it has been shown that the anion MoO₄⁼ gave a strong film condensation of didodecanoyl-LPG monolayers at pH 6 corresponding to a change in the bulk morphology from a transparent gel to a lamellar liposomal structure. A similar decrease in the molecular packing of S. aureus LPG was induced by MoO₄⁼, without a change in the freeze etch morphology of the dispersion. Evidence is presented on the important role that the polar head groups of these charged phospholipids and their ionic environment have on the overall molecular packing. Differential scanning calorimetric measurements demonstrated that the liquid-crystalline to gel transition of didodecanoyl-PG is strongly dependent on the cations in the suspension. These phenomena may be relevant to the physical state of lipids in biological membranes.     

Lipid monolayers: Ionic impurities and their influence on the surface potentials of neutral phospholipids

The ionic effects attributed to lecithin and sphingomyelin are due to ionic contaminants present in ordinary preparations of these lipids. Removal of acidic impurities by silic acid chromatography produces lecithins which are neutral and do not show interaction with Ca⁺⁺ by surface potential measurements. Similarly, improved preparations of sphingomyelin show a zero charge, whereas silicic acid chromatography removes cationic impurities which otherwise confer sphingomyelin a positive charge.     

The active transport of Mg++ and Mn++ into the yeast cell

Certain bivalent cations, particularly Mg⁺⁺ and Mn⁺⁺, can be absorbed by yeast cells, provided that glucose is available, and that phosphate is also absorbed. The cation absorption is stimulated by potassium in low concentrations, but inhibited by higher concentrations. From the time course studies, it is apparent that the absorption rather than the presence of phosphate and the potassium is the important factor. Competition studies with pairs of cations indicate that binding on the surface of the cell is not a prerequisite to absorption. The absorption mechanism if highly selective for Mg⁺⁺ and Mn⁺⁺, as compared to Ca⁺⁺, Sr⁺⁺, and UO₂⁺⁺, whereas the binding affinity is greatest for UO₂⁺⁺, with little discrimination between Mg⁺⁺, Ca⁺⁺, Mn⁺⁺, and Sr⁺⁺. In contrast to the surface-bound cations which are completely exchangeable, the absorbed cations are not exchangeable. It is concluded that Mg⁺⁺ and Mn⁺⁺ are actively transported into the cell by a mechanism involving a phosphate and a protein constituent.     

The Permeability of the Sodium Channel to Metal Cations in Myelinated Nerve

The relative permeability of sodium channels to eight metal cations is studied in myelinated nerve fibers. Ionic currents under voltage-clamp conditions are measured in Na-free solutions containing the test ion. Measured reversal potentials and the Goldman equation are used to calculate the permeability sequence: Na⁺ ≈ Li⁺ > Tl⁺ > K⁺. The ratio P_K/P_Na is 1/12. The permeabilities to Rb⁺, Cs⁺, Ca⁺⁺, and Mg⁺⁺ are too small to measure. The permeability ratios agree with observations on the squid giant axon and show that the reversal potential E_Na differs significantly from the Nernst potential for Na⁺ in normal axons. Opening and closing rates for sodium channels are relatively insensitive to the ionic composition of the bathing medium, implying that gating is a structural property of the channel rather than a result of the movement or accumulation of particular ions around the channel. A previously proposed pore model of the channel accommodates the permeant metal cations in a partly hydrated form. The observed sequence of permeabilities follows the order expected for binding to a high field strength anion in Eisenman's theory of ion exchange equilibria.     

The Effect of Surface Charge on the Voltage-Dependent Conductance Induced in Thin Lipid Membranes by Monazomycin

Differences in the behavior of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) thin lipid membranes treated with monazomycin are shown to be due to the negative surface charge on PG membranes. We demonstrate that shifts of the conductance-voltage (g-V) characteristic of PG films produced by changes of univalent or divalent cation concentrations result from changes of the membrane surface potential on one or both sides. In particular, if divalent cations are added to the aqueous phase not containing monazomycin, the resulting asymmetry of the surface potentials results in an intramembrane potential difference not recordable by electrodes in the bulk phases. Nevertheless, this intramembrane potential difference is "seen" by the monazomycin, and consequently the g-V characteristic is shifted along the voltage axis. These changes are accounted for by diffuse double layer theory. Thus we find it unnecessary to invoke specific binding of Mg⁺⁺ or Ca⁺⁺ to the negative charges of PG membranes to explain the observation that concentrations of these ions some 100-fold lower than that of the univalent cation present produce large shifts of the g-V characteristic. We suggest that analogous shifts of g-V characteristics in axons produced by changes of divalent cation concentration are also best explained by diffuse double layer theory.     

Dielectric saturation of the aqueous boundary layers adjacent to charged bilayer membranes

Summary The surface charge density resulting from the adsorption of hydrophobic anions of dipicrylamine onto dioleyl-lecithin bilayer membranes has been measured directly using a high field pulse method. The surface charge density increases linearly with adsorbate concentration in the water until electrostatic repulsion of impinging hydrophobic ions by those already adsorbed becomes appreciable. Then Gouy-Chapman theory predicts that surface charge density will increase sublinearly, with the power [z ⁺/(z ⁺+2)] of the adsorbate concentration, wherez ⁺ is the cation valence of the indifferent electrolyte screening the negatively charged membrane surface. The predicted 1/3 and 1/2 power laws for univalent and divalent cations, respectively, have been observed in these experiments using Na⁺, Mg⁺⁺, and Ba⁺⁺ ions. Gouy-Chapman theory predicts further that the change from linear to sublinear dependence takes place at a surface charge density governed by the static dielectric constant of water and the concentration of indifferent electrolyte. Quantitative agreement with experiment is obtained at electrolyte concentrations of 10^–4 m and 10^–3 m, but can be maintained at higher concentrations only if the aqueous dielectric constant is decreased. A transition field model is proposed in which the Gouy-Chapman theory is modified to take account of dielectric saturation of water in the intense electric fields adjacent to charged membrane surfaces.     

Effect of surface potential on the intramembrane electrical field measured with carotenoid spectral shift in chromatophores from Rhodopseudomonas sphaeroides

Changes in the surface potential, the electrical potential difference between the membrane surface and the bulk aqueous phase were measured with the carotenoid spectral shift which indicates the change of electrical field in the membrane. Chromatophores were prepared from a non-sulfur purple bacterium, Rhodopseudomonas sphaeroides, in a low-salt buffer. Surface potential was changed by addition of salt or by pH jump as predicted by the Gouy-Chapman diffuse double layer theory.When a salt was added at neutral pH, the shift of carotenoid spectrum to shorter wavelength, corresponding to an increase in electrical potential at the outside surface, was observed. The salts of divalent cations (MgSO₄, MgCl₂, CaCl₂) were effective at concentrations lower than those of monovalent cation salts (NaCl, KCl, Na₂SO₄) by a factor of about 50. Among the salts of monoor divalent cation used, little ionic species-dependent difference was observed in the low-concentration range except that due to the valence of cations. The pH dependence of the salt-induced carotenoid change was explained in terms of the change in surface charge density, which was about 0 at pH 5–5.5 and had negative values at higher pH values. The dependence of the pH jump-induced absorbance change on the salt concentration was also consistent with the change in the charge density. The surface potential change by the salt addition, which was calibrated by H⁺ diffusion potential, was about 90 mV at the maximum. From the difference between the effective concentrations with salts of mono- and divalent cations at pH 7.8, the surface charge density of (?1.9 ± 0.5) · 10^?3 elementary charge per Ų, and the surface potential of about ?100 mV in the presence of about 0.1 mM divalent cation or 5 mM monovalent cation were calculated.     

Cation-induced aggregation of membrane vesicles isolated from vascular smooth muscle

Cations stimulated aortic muscle membrane aggregation with increasing potency according to their effective charge, e.g., K⁺2+3+, and the stimulation is reciprocally related to the apparent affinity for these cations. Divalent metal ion-induced membrane aggregation showed a dependence on the ionic radius, being optimal for Cd²⁺. Polyvalent cation-induced membrane aggregation was reversibly suppressed by high ionic strength as well as by metal ion chelators, irreversibly inhibited by the cross-linking agent glutaraldehyde, and enhanced by increasing concentrations of ethanol and increased temperature of the medium. When the pH is lowered below 6.0, membrane aggregation progressively increased with a concomitant decrease in cation-induced aggregation. The patterns of aggregation of microsomal membranes and further purified plasma membranes were almost identical whereas the aggregation of the heterogeneous mitochondrial membrane-enriched fraction was distinctly different in the initial rate of aggregation, its pH dependence, and metal ion concentration dependence. Our results indicate that cation-induced membrane aggregation can also be used to isolate a plasma membrane-enriched fraction from vascular smooth muscle.     

Voltage-Dependent Conductance Induced in Thin Lipid Membranes by Monazomycin

When present in micromolar amounts on one side of phospholipid bilayer membranes, monazomycin (a positively charged, polyene-like antibiotic) induces dramatic voltage-dependent conductance effects. Voltage clamp records are very similar in shape to those obtained from the potassium conductance system of the squid axon. The steady-state conductance is proportional to the 5th power of the monazomycin concentration and increases exponentially with positive voltage (monazomycin side positive); there is an e-fold change in conductance per 4–6 mv. The major current-carrying ions are univalent cations. For a lipid having no net charge, steady-state conductance increases linearly with KCl (or NaCl) concentration and is unaffected by Ca⁺⁺ or Mg⁺⁺. The current-voltage characteristic which is normally monotonic in symmetrical salt solutions is converted by a salt gradient to one with a negative slope-conductance region, although the conductance-voltage characteristic is unaffected. A membrane treated with both monazomycin and the polyene antibiotic nystatin (which alone creates anion-selective channels) displays bistability in the presence of a salt gradient. Thus monazomycin and nystatin channels can exist in parallel. We believe that many monazomycin monomers (within the membrane) cooperate to form a multimolecular conductance channel; the voltage control of conductance arises from the electric field driving monazomycin molecules at the membrane surface into the membrane and thus affecting the number of channels that are formed.     

Computation of the Electrical Double Layer Properties of Semipermeable Membranes in Multicomponent Electrolytes

A methodology is presented for calculating of the surface potential, Donnan potential, and ion concentration profiles for semipermeable microbial membranes that is valid for an arbitrary electrolyte composition. This model for surface potential, Donnan potential, and charge density was applied to recently reported experimental data for gram-positive bacteria, including Bacillus brevis, Rhodococcus opacus, Rhodococcus erythropolis, and Corynebacterium species. These calculations show that previously unconsidered trace amounts of divalent and trivalent cations at very low concentrations (10⁻⁶ M) can have significant effects on the calculated surface and Donnan potentials, at ionic strengths of I ≤ 0.01 M, and that these effects need to be considered in accurate modeling of microbial surface. In addition, the calculated ion concentration profiles show that owing to the relatively high surface charges that can develop in microbial membranes, electrostatic effects can act to significantly concentrate divalent (factors of 5 × 10³) and trivalent (factors of 2 × 10⁴) cations within the bacterial cell wall. Comparison of the calculated concentration factors with those derived from experiments shows that a significant fraction of the uptake of metal by bacteria can be explained by the proposed electrostatic model.     

Triple helix formation and disulphide bonding during the biosynthesis of glomerular basement membrane collagen.

White erythrocyte membranes, or ghosts, were monoconcave discocytes when incubated in 50mM N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid titrated to pH 7.4 with triethanolamine. If 3mM MgCl₂ was included in the incubation medium, the ghosts were predominantly echinocytes. The echinocytic form could also be induced by Co⁺⁺, Ni⁺⁺, Li⁺, Na⁺, K⁺, $\text{NH}{}_4{}^{\mathrm{+}}$ and tetramethylammonium ion, all as chloride salts. The concentration of cation necessary for 50% of the ghosts to be echinocytes was correlated with the hydrated charge density of the cation with the most highly charged cations being the most effective. The cations Ca⁺⁺, Sr⁺⁺, Ba⁺⁺ and La⁺⁺⁺, (also as chloride salts) did not induce the normal echinocytic form, but at high levels induced a few misshapen forms with some resemblance to echinocytes. Instead Ca⁺⁺, Sr⁺⁺, Ba⁺⁺ and La⁺⁺⁺ suppressed the formation of echinocytes in the presence of Mg⁺⁺ and other ions. This suggests the presence of a specific Ca⁺⁺ binding site important to shape control in the erythrocyte membrane.     

Bioelectric effects of ions microinjected into the giant axon of Loligo

1. A technique is described for recording the bioelectric activity of the squid giant axon during and following alteration of the internal axonal composition with respect to ions or other substances. 2. Experimental evidence indicates that the technique as described is capable of measuring changes in local bioelectric activity with an accuracy of 10 to 15 per cent or higher. 3. Alterations of the internal K⁺ or Cl^- concentrations do not cause the change in resting potential expected on the basis of a Donnan mechanism. 4. The general effect of microinjection of K⁺ Rb⁺, Na⁺, Li⁺, Ba⁺⁺, Ca⁺⁺, Mg⁺⁺, or Sr⁺⁺ is to cause decrease in spike amplitude, followed by propagation block. 5. The resting potential decreases when the amplitude of the spike becomes low and block is incipient. 6. The decrease in resting potential and spike amplitude may be confined to the immediate vicinity of the injection. 7. At block, the resting potential decreases up to 50 per cent, but injection of small quantities of divalent cations may cause much larger localized depolarization. 8. The blocking effectiveness of K⁺, Na⁺, and Ca⁺⁺ expressed as reciprocals of the relative amounts needed to cause block is approximately 1:5:100. Rb⁺ has the same low effectiveness as does K⁺. Li⁺ resembles Na⁺. Ba⁺⁺ and Mg⁺⁺ are approximately as effective as Ca⁺⁺. 9. Microinjection of Na⁺ may cause marked prolongation of the spike at the injection site as well as decrease in its amplitude. 10. The anions used (Cl^-, HCO₃^-, NO₃^-, SO₄^-, aspartate, and glutamate) do not seem to exert specific effects. 11. A tentative explanation is offered for the insensitivity of the resting potential to changes in the axonal ionic composition. 12. New data are presented on the range of variation, in a large sample, of the magnitude of the resting potential and spike amplitude.     

Electrical stability of erythrocytes in the presence of divalent cations

Erythrocytes suspended in a medium of low ionic strength lyse under the effect of an exponential electrical pulse. The percentage of haemolysed cells decreases several-fold in the presence of divalent cations. The protective action of the ions studied increases in the following order: Ca⁺⁺, Mg⁺⁺, Zn⁺⁺. It is assumed that divalent ions bind to the negative charges of the lipid and protein molecules and reduce their electrostatic repulsion, which results in stabilization of the membranes.     

Effect of magnesium and calcium ions on blocking of synaptic transmission by adenosine in isolated rat hippocampal slices

Dependence of the inhibitory action of adenosine on the extracellular composite EPSP on the concentrations of Mg and Ca cations in the medium was investigated in isolated slices of rat hippocampusin vitro. Extracellular EPSPs were derived in the region of apical dendrites of pyramidal cells in area CA₁ during stimulation of Schaffer's collaterals. The blocking action of bivalent cations (an increase in Mg⁺⁺ or a decrease in Ca⁺⁺) developed almost five times more slowly than the action of adenosine. An increase in the external Mg⁺⁺ concentration potentiated whereas a decrease weakened the inhibitory action of adenosine. Ca⁺⁺ ions had the opposite effect. Antagonistic relations were exhibited between Mg⁺⁺ and Ca⁺⁺ ions. Analysis of dose-response curves for adenosine showed that during a simultaneous increase in the extracellular Ca⁺⁺ and decrease in Mg⁺⁺ concentrations, not only was the maximal effect of adenosine reduced, but so also was its binding constant with the receptor. The results suggest that antagonism between Ca cations and adenosine is mixed in character — both competitive and noncompetitive. The possible mechanism of the inhibitory action of adenosine on synaptic transmission and the role of bivalent cations in this process are discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 4, pp. 532–539, July–August, 1984.     

Polarographic investigation of binding of Cu++ and Cd++ by DNA

The equilibrium binding constants of Cd⁺⁺ and Cu⁺⁺ to native and denatured calf thymus DNA were determined polarographically. The binding constants are an exponential function of the potential at the binding site and as such they vary with ionic strength and with the charge on the DNA molecule. The correlation between the fraction of sites occupied by heavy metal ions and between the thermal stability of DNA in solution is discussed.     

Mechanism and Role of Divalent Cation Binding of Bacteriorhodopsin

Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na⁺ and K⁺), or trivalent, (La⁺⁺⁺) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La⁺⁺⁺. The kinetics of the M photointermediate and light-induced H⁺ uptake are not affected by Na⁺ and K⁺, but they are drastically lengthened by La⁺⁺⁺ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La⁺⁺⁺-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin.     

The effect of multivalent cations on the membrane potential of the Ehrlich ascites tumor cell

The effect of di- and trivalent cations on the membrane potential of the Ehrlich ascites tumor cell has been investigated using micro-electrode techniques. In solutions free of multivalent cations the average membrane potential for 46 cells was 8.3 ± 0.5 mv (SE). However, the potentials were not stable and decayed with a half-time of about six seconds. Addition of Ca⁺⁺ decreased the rate of decay and concomitantly increased the membrane potential. The magnitude of these effects was a function of the Ca⁺⁺ concentration. At the optimum concentration (2 mM ), the half-time of decay was increased to 12 seconds and the membrane potential was raised to 17.8 ± 1.7 mv (SE). The related alkaline-earth cations, Sr⁺⁺, Ba⁺⁺ and Mg⁺⁺ had similar effects on both the stability and magnitude of the membrane potential. The effect of La⁺⁺⁺, which was qualitatively similar to that of the divalent cations, was also concentration dependent. However, 100-fold lower concentrations were adequate to achieve comparable effects. Moreover, membrane potentials were stable for up to ten minutes in La⁺⁺⁺-containing solutions. Variations in intracellular Cl^? content induced by temperature changes were paralleled by changes in membrane potentials. However, the potentials were not those expected for a simple Cl^? electrode.     

Perturbational effects of inorganic cations on human erythrocyte membranes

Summary The perturbational effects of monovalent and divalent cations on human erythrocyte membranes were analyzed by examining their influence on kinetic and structural characteristics of trinitrobenzenesulfonic acid (TNBS) incorporation into the amino groups of protein and phospholipid structural components. The stimulatory effects of monovalent cations on TNBS incorporation, which were size-independent and attributed to nonspecific membrane alterations resulting from ionic strength factors, contrasted with the more pronounced stimulatory properties of divalent cations which were markedly size-dependent. These stimulatory effects of cations on TNBS incorporation were associated with alterations not only in rate but also in activation energy of incorporation. Changes in activation energy produced by divalent cations paralleled their ability to perturb membrane protein components and probably reflected changes in probe permeation. The rate of TNBS incorporation exhibited a dependence on divalent cation ionic radius which paralleled ion-induced perturbations in the labelling of the membrane amino phospholipid phosphatidylethanolamine. Divalent cations differed both in the relative extent and in the characteristics of protein and phospholipid perturbation. Alkaline earth cations behaved as a rather homogeneous group while Ni⁺⁺, Co⁺⁺ and Mn⁺⁺ constituted a second heterogeneous group. The influence of monovalent and divalent cations on the hemolytic behavior of intact erythrocytes paralleled their effects on TNBS incorporation into isolated membranes rather closely. It is suggested that TNBS incorporation may provide a valuable means of analyzing functionally relevant cation-induced alterations in biological membranes in general.     

Comparative Study of the Condensation of Chicken Erythrocyte and Calf Thymus Chromatins by Di- and Multivalent Cations

Abstract

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidityJulprecipitation and electric dichroism measurements. For all the cations investigated (Mg²⁺, Tb³⁺, Co(NH₃)₆ ³⁺, spermidine Spd²⁺ and spermine Sp⁴⁺) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg²⁺ and Tb³⁺. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a “precondensed” state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the “cross-linker” models. Tb³⁺ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that “30 nm fibers” were formed. Very little aggregation was produced by Tb³⁺. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.