pH gradient across the lysosomal membrane generated by selective cation permeability and Donnan equilibrium.

The pH within isolated Triton WR 1339-filled rat liver lysosomes was determined by measuring the distribution of [14C]methylamine between the intra- and extralysosomal space. The intralysosomal pH was found to be approximately one pH unit lower than that of the surrounding medium. Increasing the extralysosomal cation concentration lowered the pH gradient by a cation exchange indicating the presence of a Donnan equilibrium. The lysosomal membrane was found to be significantly more permeable to protons than to other cations. The relative mobility of cations through the lysosomal membrane is H+ greater than Cs+ greater than Rb+ greater than K greater than Na+ greater than Li+ greater than Mg2+, Ca2+. The presented data suggest that the acidity within isolated Triton WR 1339-filled lysosomes is maintained by: (1) a Donnan equilibrium resulting from the intralysosomal accumulation of nondiffusible anions and (2) a selective permeability of the lysosomal membrane to cations.     

Extent of charge screening in aqueous polysaccharide solutions

The absorption optical system of a Beckman XL-I ultracentrifuge has been used to monitor the Donnan distribution of ions in polysaccharide solutions dialyzed against sodium phosphate buffer (pH 6.8, I 0.08) supplemented with 0.2 mM chromate as an indicator ion. For dextran sulfate, heparin, and polygalacturonate, the effective net charges are shown to be only one-third of those deduced from the chemical structures--a reflection of charge screening (counterion condensation) in aqueous polyelectrolyte solutions. Whereas the extent of charge screening for the first two polysaccharides agrees well with theoretical prediction, the disparity in the corresponding comparison for polygalacturonate reflects partial esterification of carboxyl groups, whereupon the experimental parameter refers to the effective charge per hexose residue rather than the effective fractional charge of each carboxyl group.     

Ionic regulation of cyclic AMP levels in Paramecium tetraurelia in vivo

cAMP levels in Paramecium increased dose dependently after a step increase of [Ca] or [Sr] in the incubation, provided K was present. Two mM Ca or Sr tripled cAMP concentrations within 3 s and induced an increase in forward swimming speed. The increase in cAMP formation was strictly dependent on the Donnan ratio [K]: square root [Ca]. Na, Li, or tetraethylammonium could not replace K. The data provide evidence for regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the regulation of cAMP in Paramecium by the membrane surface charge as determined specifically by the K: Ca ratio.     

Induction of Na+ channel voltage sensitivity in Xenopus oocytes depends on Ca2+ mobilization

An unusual inward current which is slowly elicited in the Xenopus oocyte membrane during sustained depolarization is reportedly carried by Na+. It is thought that Na+ selective channels are in some way induced to become voltage-sensitive by the depolarization. Earlier studies report that the induction process involves a phospholipase C and a protein kinase C as well as calcium ions. The present work investigated the origins of this calcium in the oocyte. We show that injection of the powerful Ca2+ chelator (BAPTA) in the oocyte, before induction of the Na+ channels, prevented the appearance of the Na+ current, confirming an important role for [Ca2+]i. However, in oocytes perfused with Ca2+ -free medium, induction of the channels could still be obtained, indicating that induction did not depend upon the entry of external Ca2+. Downmodulation of Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores with caffeine and with a low molecular weight heparin resulted in decreased or no Na+ currents. The results are discussed in terms of the contributions from other endogenous calcium-dependent conductances which can influence the Na+ current amplitudes and time courses. The results presented support the idea that intracellular Ca2+ increase principally due to Ca2+ released from InsP3-sensitive stores is needed by the enzyme systems to produce the depolarization-induced activation of the Na+ conductance in the Xenopus oocyte.     

A mathematical model of blood-interstitial acid-base balance: application to dilution acidosis and acid-base status

We developed mathematical models that predict equilibrium distribution of water and electrolytes (proteins and simple ions), metabolites, and other species between plasma and erythrocyte fluids (blood) and interstitial fluid. The models use physicochemical principles of electroneutrality in a fluid compartment and osmotic equilibrium between compartments and transmembrane Donnan relationships for mobile species. Across the erythrocyte membrane, the significant mobile species Cl? is assumed to reach electrochemical equilibrium, whereas Na(+) and K(+) distributions are away from equilibrium because of the Na(+)/K(+) pump, but movement from this steady state is restricted because of their effective short-term impermeability. Across the capillary membrane separating plasma and interstitial fluid, Na(+), K(+), Ca2(+), Mg2(+), Cl?, and H(+) are mobile and establish Donnan equilibrium distribution ratios. In each compartment, attainment of equilibrium by carbonates, phosphates, proteins, and metabolites is determined by their reactions with H(+). These relationships produce the recognized exchange of Cl(-) and bicarbonate across the erythrocyte membrane. The blood submodel was validated by its close predictions of in vitro experimental data, blood pH, pH-dependent ratio of H(+), Cl?, and HCO?? concentrations in erythrocytes to that in plasma, and blood hematocrit. The blood-interstitial model was validated against available in vivo laboratory data from humans with respiratory acid-base disorders. Model predictions were used to gain understanding of the important acid-base disorder caused by addition of saline solutions. Blood model results were used as a basis for estimating errors in base excess predictions in blood by the traditional approach of Siggaard-Andersen (acid-base status) and more recent approaches by others using measured blood pH and Pco? values. Blood-interstitial model predictions were also used as a basis for assessing prediction errors of extracellular acid-base status values, such as by the standard base excess approach. Hence, these new models can give considerable insight into the physicochemical mechanisms producing acid-base disorders and aid in their diagnoses.     

Thallium and cesium in muscle cells compete for the adsorption sites normally occupied by K+

It was shown that Tl+ can stoichiometrically displace K+ in frog muscle cells. This displacement is independent of the function of cell membrane and postulated pumps and of a macroscopic electric charge or Donnan effect. It follows that Cs+ and Tl+ compete for the adsorption sites normally occupied by K+. On this basis, the association-induction hypothesis predicts that Cs+ and Tl+, like K+, should be primarily localized in the A-band of the muscle myofibrils.     

Donnan effect as measured by sedimentation equilibrium for the protein cytochrome c.

The protein turkey-heart cytochrome c is used as a model protein to study charge effects in sedimentation equilibrium experiments in three-component solutions. Data are given for the dependence of the apparent M (1–υ ρ) on ρ in solutions of KCl, RbCl, CsCl, and triethylamine hydrochloride. The results show the Donnan effect to have a significant influence on the apparent molecular weight, found by extrapolation of the data to a solution density of one. The apparent molecular weights are for protein at infinite dilution. A theoretical treatment is presented where the magnitude of this effect can be predicted accurately from the formal net charge of the protein as computed from the amino acid composition. The results are shown to be important in computing the preferential hydration of the protein in concentrated salt solutions. For such systems the Donnan effect should be subtracted from the total interaction coefficient for multicomponent system in order to obtain the preferential hydration.     

A new radiochemical method to investigate ion binding with polyelectrolytes

A new method for investigating the binding of ions with polyelectrolytes has been developed. This method, based on Donnan equilibrium and an isotope exchange between the electrolyte and polyelectrolyte, can distinguish territorial from specific binding of ions and can determine fractions of ions bound with the polyion. This method can determine ion binding with polyelectrolytes in a wide range of polyelectrolyte concentrations in multicomponent solutions. The method was tested with radioactive tracers 22Na+, 36Cl- and heparin sodium salt. The influence of the ionic strength on the Na+ binding with heparin was investigated at 310 K. In the limit of zero ionic strength, all Na+ ions are bound to heparin, but only 45% of them are exchangeable. Thus Na+ ions can be bound both territorially and specifically. The fraction of bound ions decreases rapidly with increasing ionic strength. The fraction of the specifically bound ions becomes negligible when the ionic strength exceeds 0.01 M, whereas the fraction of territorially bound ions can be neglected at ionic strengths higher than 0.45 M.     

The relationship between the poisson-boltzmann model and the condensation hypothesis: an analysis based on the low salt form of the Donnan coefficient

Two common models for the interaction of counterions with cylindrical polyions are considered in the context of the Donnan membrane equilibrium. General analytic expressions are obtained from the Poisson-Boltzmann equation for the Donnan coefficient in terms of the potential at the surface of the polyion or the local concentration of unbound ions at the surface. Analysis based on these expressions shows that if, and only if, the polyion charge density exceeds a certain critical value a large local concentration of ions will persist near the polyion surface at low ionic strengths. We therefore conclude that this principal hypothesis of the condensation model is consistent with the characteristics of the Poisson-Boltzmann potential at the surface of the polyion.     

23Na-nuclear magnetic resonance study of ionophore-mediated cation exchange between two populations of liposomes.

A model system to observe and investigate the transfer of Na+ ions between different internal compartments in suspension of liposomes was developed, and the exchange was followed by nuclear magnetic resonance spectroscopy. The experiments were performed under conditions of a Donnan equilibrium. Quantitative analysis of this three-site transmembrane exchange system allowed us to distinguish between direct and indirect exchange between liposomes. It also disclosed a "confining" effect on the exchange between the two populations of liposomes. This confining effect may have been due to an electrostatic field in the presence of a membrane potential. Donnan potentials and ionic compositions at equilibrium for the three-compartment system were calculated numerically. The model system may be used to explore further the effects of membrane potentials, surface potentials, and ionic mobilities on ion transport in biological (model) systems in general.     

Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Monovalent ion current through single calcium channels of skeletal muscle transverse tubules.

Akali monovalents, Li, Na, K, Cs, and organic monovalents of molecular cross section less than 20 A2, ammonium, methylammonium, hydrazinium, guanidinium, are shown to have a measurable conductance through Ca channels of muscle transverse tubules reconstituted into planar bilayers. For the alkali series, single channel conductances follow the sequence Cs approximately equal to K greater than Na greater than Li with a conductance ratio [g(Cs)/g(Li)] = 1.7. For permeability ratios, the sequence is Li greater than Na greater than K approximately equal to Cs with [P(Li)/P(Cs)] = 1.5. Monovalent current is only unmasked when Ba ions are not present. In mixtures of Cs and Ba, single channel current reverses close to the Ba equilibrium potential and more than 100 mV away from the Cs equilibrium potential. A cutoff in conduction is found for organic cations larger than trimethylammonium; this suggests an apparent pore aperture of about 5 X 5 A. Even in such a large pore, the fact that the alkali cation permeability sequence and conductance sequence are inverted rules out molecular sieving as the mechanism of selection among monovalents.     

The effect of counter-ion substitution on the transport properties of polyelectrolyte solutions

The effects of counter-ion substitution in aqueous polyelectrolyte solutions (chondroitin sulfate) on the two main transport phenomena of the ionic species, self-diffusion and electrical mobility, were studied experimentally by tracer methods and dynamic light scattering. The data were analyzed with respect to counter-ion condensation and stoichiometric substitution of low-ionic counterions by high-ionic charge ones and compared to Manning's theory. Substitution effects on the apparent charge of the macro-ion were derived from the transport data using an extended Nernst-Einstein relationship and discussed in the light of the condensation effect in polyelectrolyte solutions. The effective charge of the polyion (i.e., its residual charge after condensation of counter-ions) and the charge difference between the substituting counter-ions appear determinant in the mechanism of substitution.     

MEMBRANE EQUILIBRIA AND THE ELECTRIC CHARGE OF RED BLOOD CELLS

It has been shown, within the probable limit of error of the methods of measurement employed, that the Donnan equilibrium determines the distribution of H and Cl ions between the cell and the surrounding fluid. This equilibrium is a consequence of the impermeability of the cell membrane to the inorganic cations of the cell. The mechanism responsible for this equilibrium is suggested as that concerned in the secretion of HCl by the cells of the gastric mucosa. If the salt concentration of the medium is low there may result from the Donnan equilibrium a thermodynamic P.D. of considerable magnitude. In the presence of low concentrations of electrolytes, this P.D. is to be regarded as positive in sign at reactions of the medium at which the cataphoretic charge of the cell is negative in sign. The explanation of this discrepancy in sign of charge may lie in the existence at an outer phase-boundary of a second Donnan equilibrium the nature of which is determined by the ionization of the protein of the cell membrane.     

Charge patch attraction and reentrant condensation in DNA-liposome complexes

We investigated the formation of complexes between cationic liposomes built up by DOTAP and three linear anionic polyions, with different charge density and flexibility, such as a single-stranded ssDNA, a double-stranded dsDNA and the polyacrylate sodium salt [NaPAA] of three different molecular weights. Our aim is to gain further insight into the formation mechanism of polyion-liposome aggregates of different sizes (lipoplexes), by comparing the behavior of DNA with a model polyelectrolyte, such as NaPAA, with approximately the same charge density but with a higher flexibility. We employed dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements, in order to explore both the hydrodynamic and structural properties of the aggregates resulting from polyion-liposome interaction and to present a comprehensive picture of the complexation process. The phenomenology can be summarized in a charge ratio-dependent scenario, where the main feature is the formation of large equilibrium clusters due to the aggregation of intact polyion-coated vesicles. At increasing polyion-liposome ratio, the size of the clusters continuously increases, reaching a maximum at a well-defined value of this ratio, and then decreases ("reentrant" condensation). The aggregation mechanism and the role of the polyion charge density in the complex formation are discussed in the light of the recent theories on the correlated adsorption of polyelectrolytes at charged interfaces. Within this framework, the phenomena of charge inversion and the reentrant condensation, peaked at the isoelectric point, finds a simple explanation.     

Electrical potentials of plant cell walls in response to the ionic environment

Electrical potentials in cell walls (psi(Wall)) and at plasma membrane surfaces (psi(PM)) are determinants of ion activities in these phases. The psi(PM) plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of psi(Wall). psi(Wall) from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of psi(Wall) with effectiveness in the order Al(3+) > La(3+) > H(+) > Cu(2+) > Ni(2+) > Ca(2+) > Co(2+) > Cd(2+) > Mg(2+) > Zn(2+) > hexamethonium(2+) > Rb(+) > K(+) > Cs(+) > Na(+). This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of psi(Wall), and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for psi(Wall) were in close agreement, usually, and we consider psi(Wall) to be at least proportional to the actual Donnan potentials. psi(Wall) and psi(PM) display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. psi(Wall) is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion.     

Potential and K+ activity in skinned muscle fibers. Evidence against a simple Donnan equilibrium

It has been suggested that potentials measured with conventional microelectrodes in chemically or mechanically skinned muscle fibers arise from a Donnan equilibrium due to myofilament fixed charges. This hypothesis was tested in mechanically skinned frog (Rana pipiens) semitendinosus fibers by measuring the distribution potential (Ed) between fiber and bath with 3 M KCl-filled microelectrodes and the K+ activity gradient (aik/aok) with K+ ion-selective microelectrodes (KISE). If skinned fibers are a Donnan system, Ed should become more positive as pH is decreased, altering the fixed charge on the myofilaments. Consistent with this expectation, Ed was -4.4, -0.6, and +4.8 mV in ATP-containing solutions and -6.5, -2.2, and +8.4 mV in ATP-free solutions at pH 7, 6, and 5, respectively. Donnan equilibrium also requires that all mobile ionic species be in electrochemical equilibrium. In ATP-containing solutions, this was true for K+ at pH 7. At pH 5, however, KISE indicated that K+ was not in equilibrium; average Ed was 5.9 mV positive to the K+ equilibrium potential, and aik/aok was 1.04, while the Donnan prediction was 0.83. In contrast, KISE measurements in ATP-free solutions indicated that K+ was in equilibrium at all pH studied. Skinned fibers in ATP-containing media are not equilibrium systems because ATPase reactions occur. Under our conditions, frog myofibrils hydrolyze 0.4 and 0.08 mumol ATP/min X mg myofibrillar protein at pH 7 and 5, respectively. It is suggested that in the presence of ATP, Ed is a superposition of Donnan and diffusion potentials, the latter arising from differences in the mobilities of anionic substrate and products that diffuse through the charged myofilament lattice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Depolarization of the membrane potential by hyaluronan

The membrane potential is mainly maintained by the K⁺ concentration gradient across the cell membrane between the cytosol and the extracellular matrix. Here, we show that extracellular addition of high‐molecular weight hyaluronan depolarized the membrane potential of human fibroblasts, human embryonic kidney cells (HEK), and central nervous system neurons in a concentration‐dependent manner, whereas digestion of cell surface hyaluronan by hyaluronidase caused hyperpolarization. This effect could not be achieved by other glycosaminoglycans or hyaluronan oligosaccharides, chondroitin sulfate, and heparin which did not affect the membrane potential. Mixtures of high‐molecular weight hyaluronan and bovine serum albumin had a larger depolarization effect than expected as the sum of both individual components. The different behavior of high‐molecular weight hyaluronan versus hyaluronan oligosaccharides and other glycosaminoglycans can be explained by a Donnan effect combined with a steric exclusion of other molecules from the water solvated chains of high‐molecular weight hyaluronan. Depolarization of the plasma membrane by hyaluronan represents an additional pathway of signal transduction to the classical CD44 signal transduction pathway, which links the extracellular matrix to intracellular metabolism. J. Cell. Biochem. 111: 858–864, 2010. © 2010 Wiley‐Liss, Inc.     

Charge formation in microporous membranes by single-acid site dissociation

The electrolyte concentration and pH dependence of the effective charge density of weak ion exchange membranes have been studied by combining solutions of the Poisson-Boltzmann equation in cylindrical pores with a simple dissociation equilibrium of weakly acid groups attached to the pore walls. Analytical expressions for the effective charge density and wall potential are presented which describe these quantities in terms of pH, electrolyte (1 : 1) concentration, acid constant, density of acid groups and the pore size. The concentration dependence of the effective fixed charge density experimentally observed for cellulose membranes and NaCl-solutions agrees quantitatively with the theoretical predictions. For track-etched mica membranes and KCl-solutions the influence of pH and electrolyte concentration on the effective charge density can be qualitatively explained. Also an interpretation of electro-osmotic findings obtained with an asymmetric cellulose acetate membrane and NaCl-solutions is given.