Mass-action formulations of monovalent and divalent cation adsorption by phospholipid membranes.

An extension of a previous treatment (Cohen, J. A., and M. Cohen, 1981, Biophys. J., 36:623-651) is presented for the adsorption of monovalent and divalent cations by single-component phospholipid membranes, where monovalent cations adsorb with a cation/phospholipid stoichiometry of 1:1 and divalent cations adsorb with stoichiometries of 1:1 and 1:2. Previously the 1:1 and 1:2 binding of divalent cations were assumed to occur by independent, parallel pathways. Here a serial adsorption scheme is considered in which 1:2 binding occurs via reaction of 1:1-bound complexes with adjacent unoccupied phospholipids. This two-dimensional lattice reaction is shown to obey a law of mass action, and the mass-action equilibrium constant is used to parameterize the adsorption isotherm. This isotherm is shown to be mathematically equivalent to the previous isotherm, although the two formulations differ in the dependence of 1:2 binding on the 1:1 association constant.     

Mono- and divalent competitive adsorption to a charged membrane in a closed system: A comparative study

Competitive adsorption to a negative surface between monovalent and divalent cations is studied in a closed system. A self-consistent theory is presented for the cases when the divalent cation binds to two negative sites (1:2 binding) and to one negative site (1:1 binding). It is demonstrated that these two cases are distinguishable when the relative difference in total divalent concentrations determined at fixed bulk divalent concentrations is plotted as a function of the bulk monovalent concentration. The 1:2 binding case yields a sigmoidal curve while the 1:1 binding curve is hyperbolic. The comparison between the 1:2 and 1:1 binding cases of the divalent cation is extended to include; (1) the existence of a surface charge to which no binding occurs, and (2) the case when an additional non-binding monovalent cation is present.     

Divalent cation-induced surface tension increase in acidic phospholipid membranes: Ion binding and membrane fusion

Chelation binding of divalent cations to phospholipid membranes may cause deformation in the headgroup regions of these lipid molecules. This deformation may be responsible for the observed large increase in surface tension of acidic phospholipid membranes induced by divalent cations. On the other hand, simple binding of monovalent cations without being followed by such a deformation of membrane molecules, does not result in a large surface tension increase in the membrane. A theoretical explanation for the above situation is given and the divalent cation-induced acidic phospholipid membrane fusion as well as other lipid membrane fusions are discussed in terms of the increased surface energy of membranes.     

Perturbational effects of inorganic cations on human erythrocyte membranes.

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 in 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.     

An energy dependent divalent cation-hydrogen ion binding in the outer membranes of rat liver mitochondria and its dependence on monovalent cation-hydrogen ion binding

Intact mitochondria are able to bind monovalent and divalent metal cations and to release protons in an energy-independent exchange process. Directly accessible binding sites exist in the outer membrane. They seem to be identical for monovalent and divalent metal ions. The inner membrane-matrix-fraction possesses exchange sites after ultrasonic disruption only for monovalent cations, but not for divalent cations.     

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.     

Ion association reactions with biological membranes,studied with the fluorescent dye 1-anilino-8-naphthalenesulfonate

Summary (1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs⁺ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs⁺>Rb⁺>K⁺>Na⁺>Li⁺. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm ^–1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.     

Divalent cation binding to phospholipids: An EPR study

Summary Divalent cation association to sonicated phospholipid liposomes has been examined with electron paramagnetic spectroscopy. Spectra were obtained suggesting that, in some cases, divalent cations associated with acidic phospholipid head groups are highly mobile.Using the amplitude of its characteristic sextet signal as a measure of free Mn(H₂O) ₆ ⁺⁺ , the apparent affinities of cardiolipin and phosphatidylserine for Mn²⁺ were measured as a function of monovalent electrolyte. Monovalent cations having smaller nonhydrated radii were more effective in displacing Mn from the phospholipids. Under conditions of low divalent cation concentrations, it is shown that the Gouy-Chapman diffuse double layer theory predicts a Mn-affinity (K _A ) inversely proportional to the square of monovalent salt concentration. Although this relationship was closely obeyed for Mn binding to cardiolipin, the fall-off inK _A with added sodium chloride was slower in the cases of Mn binding to phosphatidylserine or phosphatidic acid.When phosphatidylcholine or cholesterol was incorporated into mixed vesicles along with a fixed amount of charged phospholipid, the Mn-binding strength was roughly proportional to the weight fraction of the latter. This result is consistent with: (1) a random dispersal of lipids in the bilayer, and (2) a 1:2 divalent cation-phospholipid interaction.     

Characterization of the receptor for epidermal growth factor-urogastrone in human placenta membranes

The binding of mouse epidermal growth factor-urogastrone (EGF-URO) to membranes from term human placenta is peptide-specific, saturable (about 20 pmol of EGF-URO bound maximally/mg of protein), reversible, and of high affinity (KD about 400 pM). Optimal binding is observed at pH 7.6. At low pH (3.5 to 5.0). EGF-URO can be reversibly dissociated from the receptor; however, exposure to pH < 3 irreversibly inactivates the receptor. The binding, which does not exhibit ligand cooperativity, exhibits an association rate constant of 6.1 x 10(-4) s-1 and a dissociation rate constant of 6.1 x 10(-4) s-1. The dissociation constant determined from the rate constants, 240 pM, is in reasonable agreement with the constant estimated by equilibrium methods. Both monovalent and divalent cations augment EGF-URO binding 2- to 3-fold. Although in general, divalent cations enhance binding at lower concentrations (optimum, 5 mM) than do monovalent cations (optimum, approximately 80 mM), there is no cation-specific effect. Neither guanine nor adenine nucleotides affect EGF-URO binding. Whereas the proteolytic enzymes (trypsin, chymotrypsin, papain, and pepsin) inactivate the receptor, neuraminidase and phospholipases A2, C, and D augment EGF-URO binding. Neuraminidase increases the number of available sites without affecting ligand affinity. Wheat germ agglutinin, concanavalin A, and phytohemagglutinin all compete for the binding of EGF-URO. The data complement previous observations of EGF-URO binding obtained in intact cells and provide a basis for the solubilization, characterization, and isolation of this receptor from a rich tissue source.     

Kinetics and thermodynamics of thermal denaturation in acyl carrier protein.

The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry. Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt. NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale. However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale. These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways. Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms. Divalent cations provide stability by binding to specific sites present only in the native state.     

The Staphylococcal Pore-forming Leukotoxins Open Ca2+ Channels in the Membrane of Human Polymorphonuclear Neutrophils

The ability of leukotoxins secreted by Staphylococcus aureus to modify the permeability of the membrane of human polymorphonuclear neutrophils has been studied by spectrofluorometry and appropriate fluorescent probes. This family of bicomponent leukotoxins is constituted by, at least, three pairs of proteins: LukS-PV/LukF-PV, HlgA/HlgB, HlgC/HlgB. After binding of both components to the membrane, each pair induces influxes of divalent cations and ethidium in polymorphonuclear neutrophils, although with different intensities. The influx of divalent cations appears sooner than the influx of ethidium. The pathway for divalent cations is not permeable to monovalent cations (Na⁺, K⁺, ethidium⁺) and is blocked by Ca²⁺ channel inhibitors that do not block the fluxes of ethidium and monovalent cations. It is concluded that the leukotoxins bind to a receptor linked to a divalent cation-selective channel or to the channel itself which is activated. Then, the leukotoxins open a second pathway by insertion into the membrane and subsequent formation of aspecific pores allowing an influx of ethidium. Received: 8 May 1997/Revised: 22 December 1997     

Thermodynamic parameters for the binding of divalent cations to gramicidin A incorporated into a lipid environment by Tl-205 nuclear magnetic resonance.

Thermodynamic parameters, enthalpy and entropy, for the binding of the divalent cations, Mg+2, Ca+2, Sr+2, Ba+2, and Cd+2, to gramicidin A, incorporated into lysophosphatidylcholine, have been determined using a combination of Tl-205 nuclear magnetic resonance spectroscopy and competition binding. The binding process is thermodynamically driven by the enthalpy and not the entropy. The enthalpy values are related to the process involving the transfer of cations from an aqueous environment to an amide environment. A comparison is made between the thermodynamic parameters for the binding of monovalent and divalent cations to gramicidin A to illustrate the channel blocking ability of the divalent cations with respect to monovalent cation transport.     

The Mg,Ca, EDTA and ATP dependence of Na binding by rat liver microsomes

The role of ionic interactions in the adenosinetriphosphate (ATP) dependent Na binding by rat liver microsomes was investigated. In the concentration range of 0 to 20 mM, Mg and Ca are demonstrated to compete strongly against Na for microsome binding sites. In the presence of Ca, the nonbiological complexing agent ethylenediaminetetraacetate (EDTA) produced a marked increase in Na binding accompanied by a concomitant decrease in Ca binding. Under similar conditions ATP, which is a weaker complexing agent than EDTA, produced quantitatively smaller but qualitatively similar changes in binding. The data show that the effect of ATP on Na binding is not dependent upon the formation of a hypothetical Na binding intermediate in the hydrolysis of ATP as other investigators have postulated. Rather, the effect of ATP is demonstrated to depend upon the presence of unhydrolyzed ATP and its ability to complex divalent cations, and thereby to reduce divalent cation competition against monovalent cations for membrane binding sites.     

Kinetics of prothrombin-mediated binding of lupus anticoagulant antibodies to phosphatidylserine-containing phospholipid membranes: an ellipsometric study

Antiphospholipid antibodies interact with phospholipid membranes via lipid binding plasma proteins, mostly, prothrombin and beta(2)-glycoprotein I. Using ellipsometry, we characterized prothrombin-mediated binding of lupus anticoagulant (LA) positive IgG, isolated from patients with antiphospholipid syndrome, to phosphatidylserine (PS)-containing membranes. LA IgG did not bind to membranes in the absence of prothrombin, but addition of prothrombin resulted in high-affinity binding of prothrombin-LA IgG complexes; half-maximal binding was attained at IgG and prothrombin concentrations of 10 microg/mL and 4 nM, respectively. Adsorption to membranes containing 10-40 mol % PS revealed that membrane-bound rather than solution-phase prothrombin determines the adsorption kinetics. Depletion of prothrombin and LA IgG from the solution results in rapid desorption which is strongly inhibited by addition of prothrombin but not of LA IgG. Prothrombin-mediated adsorption of monovalent Fab1 fragments prepared from patient LA IgG was negligible, indicating that monovalent interaction between prothrombin and LA IgG is weak. The kinetics of adsorption and desorption indicate that divalent binding of LA IgG to prothrombin at the lipid membrane occurs.     

Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.     

Kinetics of Ca2+ and Sr2+ uptake by yeast. Effects of pH, cations and phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.     

Ionophore A23187: the effect of H+ concentration on complex formation with divalent and monovalent cations and the demonstration of K+ transport in mitochondria mediated by A23187.

The two-phase extraction technique has been used to study the equilibrium between A23187, metal cations, and H+. Under these conditions the ionophore forms charge neutral isostoichiometric complexes with divalent cations in which both carboxylate groups of the 2:1 A23187:M2+ complexes are deprotonated. In ethanol, however, the methyl ester of A23187 also binds divalent cations indicating that protonated complexes between A23187 and cations should also exist. With monovalent cations, A23187 forms two charge-neutral complexes of stoichiometries and relative stabilities: A2HM greater than AM. Examination of energy utilization K+ and H+ movements, and light scattering capacity of mitochondria in the presence of divalent cation chelators, A23187, and valinomycin demonstrates that A23187 can act as a nigericin type K+ ionophore under appropriate conditions. Formation constants for the A2HM complexes with monovalent cations indicate that with appropriate conditions transport of Li+ and Na+ mediated by A23187 would also be expected. The binding constant data and associated free energies of complex formation are compared as a function of ionic radius and of cation charge. The data indicate that lack of conformational mobility in A23187 is responsible for the high cation size selectivity of this compound. To explain the transport selectivity of A23187 for divalent cations, it is proposed that this ionophore forms a family of five complexes, isostoichiometric between cations of different valence but of which only charge-neutral species are permeant to membranes. The charge of a given complex is in turn determined by that of the cation. The concept is consistent with the divalent cation transport specificity of A23187, explains the observed monovalent cation transport, and is useful in rationalizing the differences in charge selectivity between A23187 and X-537A.     

Effect of inorganic cations on phase transitions.

The effect of protons and cations on the crystal (gel)-to-liquid crystal transition temperature Tm of isoelectric and negatively charged phospholipids are summarized. The general trends emerging are as follows: Tm depends on the state of ionization of the phospholipid in that Tm-vs-pH-curves parallel the titration curve of the phospholipid. Protonation of phospholipids causes Tm to increase, deprotonation or ionization has the opposite effect. The effects of cations on the Tm of phospholipids may be grouped into non-specific and specific effects. Unspecific effects of cations such as the screening of negative charges of the phospholipid polar group are qualitatively similar to protonation: Tm increases, in the order monovalent less than divalent less than trivalent cations and the effects on negatively charged phospholipids are larger than those on isoelectric phospholipids. Unspecific, electrostatic effects on Tm are reasonably well accounted for by the Gouy-Chapman theory. If, however, specific binding comes into play and/or electrostatic effects are accompanied by changes in phospholipid structure, simple, electrostatic theories fail to explain the observed changes in Tm. The crystal (gel)-to-liquid crystal transition is also a function of the degree of hydration: Tm generally decreases with increasing hydration reaching a plateau in excess H2O. In addition to screening of electric charges, ions may exert yet another non-specific effect: ions may affect Tm indirectly by competing with the phospholipid polar group for water of hydration. This indirect effect plays a role at high ionic strength and/or at low hydration of the phospholipid. Specific binding of cations to negatively charged phospholipids can lead to tight associations of the metal ion with the lipid polar group. Isothermal crystallization of the phospholipid bilayer is induced that is accompanied by a total or partial loss of water of hydration resulting in a marked increase in Tm. For instance, in crystalline Ca2(+)-phosphatidylserine complexes Tm is increased by more than 100 degrees C.     

Regulation by Cations of [3H]Spiroperidol Binding Associated with Dopamine Receptors of Rat Brain

Abstract: The effects of monovalent and divalent cations on binding of [³H]spiroperidol to dopamine receptors in rat corpus striatum were studied. Both monovalent and divalent cations as well as several chelating agents increase the number of [³H] spiroperidol binding sites. Manganese is most potent, enhancing binding at 1 μ m concentration, while magnesium and calcium are at least two orders of magnitude less potent and the monovalent cations sodium, potassium and lithium are still weaker. Divalent cations enhance the potency of dopaminergic agonists in competing for [³H]spiroperidol binding, an effect which appears to be independent of the ionic augmentation of [³H]spiroperidol binding. Divalent cations decrease both the association and dissociation rates of [³H]spiroperidol binding to dopamine receptor sites.