Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

Surface potential on the periplasmic side of the photosynthetic membrane of Rhodopseudomonas sphaeroides

Membrane surface potential on the periplasmic side of the photosynthetic membrane was estimated in cells, spheroplasts and chromatophores of Rhodopseudomonas sphaeroides. When the membrane potential (potential difference between bulk aqueous phases) was kept constant in the presence of carbonylcyanide m-chlorophenylhydrazone, addition of salt to a suspension of cells or spheroplasts induced a red shift in the carotenoid absorption spectrum which indicated a change in the intramembrane electrical field. The spectral shift is explained by a rise in electrical potential at the outside surface of the photosynthetic membrane due to a decrease in extent of the negative surface potential.The spectral shift occurred in the direction opposite to that in chromatophores, indicating that the sidedness of the membrane of cells or spheroplasts is opposite to that of chromatophores. The dependences of the extent of the potential change on concentration and valence of cations of salts agreed with the Gouy-Chapman relationship on the electrical diffuse double layer. The charge density on the periplasmic surface of the photosynthetic membrane was estimated to be ?2.9 · 10^?3 elementary charge per Ų, while that on the cytoplasmic side surface was calculated as ?1.9 · 10^?3 elementary charge per Ų (Matsuura, K., Masamoto, K., Itoh, S. and Nishimura, M. (1979) Biochim. Biophys. Acta 547, 91–102). Surface potential on the periplasmic side of the photosynthetic membrane was estimated to be about ?50 mV at pH 7.8 in the presence of 0.1 M monovalent salt.     

Surface potential and reaction of membrane-bound electron transfer components. I. Reaction of P-700 in sonicated chloroplasts with redox reagents

Salt- or pH-induced change of the rate of reduction of the phtooxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts.

The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts.

The change of the reduction rate was analyzed using the Gouy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Brönsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface.

The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.     

Surface potential and reaction of membrane-bound electron transfer components. I. Reaction of P-700 in sonicated chloroplasts with redox reagents.

Salt- or pH-induced change of the rate of reduction of the photoxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts. The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts. The change of the reduction rate was analysed using the Guoy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Br?nsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface. The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.     

On the mechanism of anion uptake by plant roots

Summary The effect of the valence of the associated cation on Cl^–-uptake by excised barley roots grown in CaSO₄ has been studied at 26°, 6° and 2°C. The uptake of Cl^– relative to that of the associated cation was found to increase in the order: trivalent > divalent > monovalent. This was explained on the expected effect of the cation on the negative charge and potential of root surfaces. A lyotropic order was observed in case of monovalent cations, whereas divalent cations showed no such order. The order observed in Cl^–-uptake from chloride solutions of monovalent cations is associated with the ability of the absorbed cation to remove Ca and Mg from the roots. Li⁺ behaved similar to divalent cations in affecting the relative Cl^–-uptake from LiCl.As to the effect of temperature on the uptake of Cl^– and associated cation, it appears that Cl^– is not taken up to any great extent at 2°C whereas cations are still adsorbed at this low temperature. This has been explained on the assumption of the presence of negative adsorption spots on the root surface which can hold cations but not anions. It appears that Cl^–-uptake by roots requires the expenditure of energy to overcome repulsion arising from the negative surface.This work is supported by AEC contract AT (11-1) — 34 project 55.     

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 concentrations 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.     

Correlation between monovalent cation-induced decreases in chlorophyll a fluorescence and chloroplast structural changes

Low concentrations (~ 3 mm) of salts of monovalent cations such as Na⁺, K⁺, and tetraethylammonium were found to decrease the turbidity of chloroplast suspensions. The turbidity changes (Δ₅₄₀) had the same kinetics, salt concentration dependence, and pH dependence as the monovalent cation-induced decreases in chlorophyll a fluorescence (9), suggesting that structural changes are the cause of the associated increases in spillover. Electron microscopy revealed that the grana are stacked when spillover is inhibited (in the absence of salts or the presence of divalent cations) and that monovalent cations cause the grana to unstack, thereby promoting spillover.     

9-Amino-acridine as a probe of the electrical double layer associated with the chloroplast thylakoid membranes

Chloroplasts washed with monovalent cations are found to quench 9-amino-acridine fluorescence after resuspension in a cation-free medium. This quenching occurs in the absence of a high energy state and can be reversed by the addition of salts. The effectiveness of these salts is related to the charge carried by the cations and appears to be essentially independent of the associated anions. The order of effectiveness is polyvalent > divalent > monovalent, and virtually no variation is found within the groups of monovalent cations and divalent cations tested. Furthermore, choline and lysine are as effective as alkali metal cations, and lysyl-lysine is almost as effective as alkaline earth metal cations. These results are consistent with an effect mediated by the electrical double layer at the membrane surface rather than chemical bonding, and can be qualitatively explained in terms of the Gouy-Chapman theory.It appears that 9-amino-acridine acts as a diffusible monovalent cation which increases its fluorescence when displaced from the diffuse layer adjacent to the negatively charged membrane surface. The 9-amino-acridine fluorescence changes have been experimentally correlated with the cation-induced chlorophyll a fluorescence changes also observed with isolated chloroplasts.     

Effect of adsorbed cations on phosphorus uptake by excised roots

Pretreatment of excised roots of Hordeum vulgare, Zea mays, and Glycine max with various salt solutions affected their subsequent rate of phosphorus absorption from 2 × 10⁻⁵m KH₂PO₄. The rate of absorption was greatest for roots pretreated with trivalent cations, intermediate with divalent cations and lowest with monovalent cations. It appeared that the pretreatment involved a rapid exchange reaction at the root surface which was reversible. A 1 min pretreatment was effective for more than 20 min. The acceleration of phosphorus uptake by roots produced by polyvalent cations may be due partly or entirely to a greater reduction in the electrical potential at the root surface or within the pores of the negatively charged cell wall by polyvalent cations than by monovalent cations.     

The surface and membrane potentials of chromatophores of photosynthetic bacteria as studied by carotenoid absorbance changes

The change in surface potential induced by addition of mono- or divalent cations to a chromatophore suspension was monitored by carotenoid absorbance changes (a probe which is intrinsic to the membrane). The change in carotenoid absorbance elicited by an alteration of the surface potential is strongly dependent on the presence of ionophores; the absorbance changes (due to addition of MgCl₂) in the presence of valinomycin or gramicidin are larger than those in the presence of carbonyl cyanide m-chlorophenylhydrazone or cabonyl cyanide p-trifluoromethoxyphenylhydrazone. These differences in carotenoid absorbance change reflect the degree in which the membrane resistance has been shunted. Gramicidin or high concentrations of valinomycin (10⁻⁶ M) appear to be sufficiently effective as shunt in order that the totality of the change in external surface potential is seen as an intramembrane potential difference as sensed by the carotenoids. It is also shown that the decay of the carotenoid changes induced by the addition of salt to the medium is a measure of the intrinsic permeability of the chromatophore membrane for the added cation.     

Effects of surface potential and membrane potential on the midpoint potential of cytochrome c-555 bound to the chromatophore membrane of Chromatium vinosum

The values of midpoint potential (Em) of cytochrome c-555 bound to the chromatophore membranes of a photosynthetic bacterium Chromatium vinosum was determined under various pH and salt conditions. After a long incubation at high ionic concentrations in the presence of carbonylcyanide m-chlorophenylhydrazone, which was added to abolish electrical potential difference between the inner and outer bulk phases of chromatophore, the Em value was almost constant at pH values between 4.0 and 8.4. With the decrease of salt concentration, the pH dependence of the Em value became more marked. Under low ionic conditions, Em became more positive with the decrease of pH. Addition of salt made the value more positive or negative at pH values higher or lower than 4.5, respectively. Divalent cation salts were more effective than monovalent cation salts in producing the positive shift of Em at pH 7.8. The Em value became more positive when the electrical potential of the inner side of the chromatophore was made more positive by the diffusion potential induced by the K+ concentration gradient in the presence of valinomycin. These results were explained by a change of redox potential at the inner surface of the chromatophore membrane, at which the cytochrome is assumed to be situated, due to the electrical potential difference with respect to the outer solution induced by the surface potential or membrane potential change. The values for the surface potential and the net surface charge density of the inner surface of the chromatophore membrane were estimated using the Gouy-Chapman diffuse double layer theory.     

Influence of monovalent and divalent cations on the surface area of phosphatidylglycerol monolayers

A theory is presented on the electrostatic properties of the surface area of phosphatidyl-glycerol monolayers spreading at an air-water interface in the presence of monovalent and divalent cations. In the present theory, the adsorption of monovalent and divalent cations to the membranes is taken into account, besides the dissociation of protons, as a possible cause of the change of surface charge density with the variation of pH or ion concentrations. It is also pointed out that, in the presence of structure-making ions such as Li⁺ and Na⁺, the nearest-neighbour interactions between proton dissociation sites become important for the monolayers in the gel state to yield a sharp expansion of the surface area with the increase of pH. The present theory shows quantitative agreements with previously-observed data.     

Divalent cation effects on membrane bending in heated erythrocytes

The thermal fragmentation of human erythrocytes involves either surface wave growth and membrane externalization at the cell rim or membrane internalization at the cell dimple. In symmetrical monovalent electrolytes an increase in membrane internalization at the cell dimple correlates with the decrease in zeta potential arising from surface charge (sialic acid residue) depletion. The influence of divalent cations on thermal fragmentation is examined in this work. The erythrocyte zeta potential decreased when divalent cations replaced some Na⁺ in the cell-suspending phase. The incidence of membrane internalization increased in rank order Ca²⁺>Ba²⁺>Mg²⁺Sr²⁺. Calcium continued to influence the thermal fragmentation of cells highly depleted of sialic acid, suggesting that the ion also interacted with membrane sites other than sialic acid. The divalent cation influence on cell fragmentation was shown to be greater than that due to zeta potential decrease alone. This conclusion was supported by the observation that the divalent cation-induced changes in zeta potential showed much less cation specificity than did the changes induced in the thermal fragmentation pattern. The result implies that the specificity of the divalent cation effects was due to interactions within the erythrocyte shear layer. The possibility that the interaction is with membrane lipids is examined.     

Modification of foliar solute concentrations by calcium in two species of wheat stressed with sodium chloride and/or potassium chloride

The effects of saline-stresses due to different salts on growth and on foliar solute concentrations in seedlings of two species of wheat that differed in salt tolerance. Triticum aestivum L. cv. Probred and Triticum turgidum L. (Durum group) cv. Aldura, were studied. Triticum aestivum is the more salt tolerant species. The salts used were NaCl, KCI, a 1:1 mixture of NaCI and KCI, and these same monovalent cation salts but mixed with CaCI₂ at a ratio of 2:1 on a molar basis of monovalent to divalent cation salts. Growth inhibition of both species was a function of media osmotic potentials. There was a small additional inhibition of growth if KCI replaced NaCI as the salinizing salt. CaCI₂ had little or no effect on growth inhibition beyond an osmotic effect except at the most severe stress level, i.e. when Ca²⁺ concentrations may be excessive. The amounts of water-soluble Ca²⁺ were about 10 times higher in leaves of plants grown in the presence of CaCI₂ than in its absence, but its concentrations even then were approximately 10% or less of those of the monovalent cations. Including CaCI₂ in growth media resulted in a reduction in the amount of Na⁺ in leaves compared to the amounts in plants grown at the same osmotic potential but in the absence of CaCI₂. Triticum aestivum was a better Na⁺-excluder than T. turgidum. With CaCI₂ in media, (Na⁺+ K⁺) remained relatively constant or increased by small amounts as media osmotic potentials décreased. In the absence of CaCI₂₊ (Na⁺+ K⁺) increased by large amounts when media osmotic potentials were at ?0.6 and ?0.8 MPa. It is concluded that the accumulation system in leaves for monovalent cations was under feed-back control, and that this control mechanism was inhibited by high media concentrations of Na⁺ and/or K⁺. Sucrose was present at a constant amount under all growth conditions. Proline started accumulating when (Na⁺+ K⁺) exceeded a threshold value of 200 μmol (g fresh weight)^?1. Its concentration was 5 to 13% of that portion of (Na⁺+ K⁺) that exceeded the threshold value.     

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 concentrations and thermodynamic activities of cations in intact Bryopsis chloroplasts

The internal cation levels of chloroplasts isolated from a green sea alga, Bryopsis maxima, were studied. Atomic absorption spectroscopy, combined with the determination of the sorbitol-impermeable and water-permeable spaces, revealed that chloroplasts contain an extremely high concentration of K⁺ and high levels of Na⁺, Mg²⁺ and Ca²⁺. A method was developed to estimate the thermodynamic activities of monovalent and divalent cations present in chloroplasts. pH changes induced by the addition of an ionophore (plus an H⁺ carrier), which makes the outer limiting membranes of chloroplasts permeable to both a cation and H⁺, were determined. Provided that the external pH was set equal to the internal pH, the internal concentration of the cation was estimated by determining the external cation concentration which gave rise to no electrochemical potential difference of the cation and hence no pH change on addition of the ionophore. The internal pH was determined by measuring distributions of radioactive methylamine and 5,5-dimethyloxazolidine-2,4-dione between the chloroplast and medium (Heldt, H.W., Werdan, K., Milovancev, M. and Geller, G. (1973) Biochim. Biophys. Acta 314, 224–241). The internal pH was also estimated by measuring pH changes caused by the disruption of the outer limiting membrane with Triton X-100. The results indicate that a significant part of the monovalent cations and most of the divalent cations are attracted into a diffuse layer adjacent to the negatively charged surfaces of membranes and proteins, or form complexes with organic and inorganic compounds present in the intact chloroplasts.     

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.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg²⁺, Ca²⁺ and Ba²⁺) and anions (SO₄²⁻ and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K⁺ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg⁺² is optimal for the physiological reaction, however, Co⁺², Mn⁺², and Ni⁺² also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.     

Cation-induced superoxide generation in tobacco cell suspension culture is dependent on ion valence

There have been many reports suggesting the involvement of reactive oxygen species (ROS), including superoxide anion (O₂^.–), in salt stress. Herein, direct evidence that treatments of cell suspension culture of tobacco (Nicotiana tabacum L.; cell line, BY‐2) with various salts of trivalent, divalent and monovalent metals stimulate the immediate production of O₂^.– is reported. Among the salts tested, LaCl₃ and GdCl₃ induced the greatest responses in O₂^.– production, whereas CaCl₂ and MgCl₂ showed only moderate effects; salts of monovalent metals such as KCl and NaCl induced much lower responses, indicating that there is a strong relationship between the valence of metals and the level of O₂^.– production. As the valence of the added metals increased from monovalent to divalent and trivalent, the concentrations required for maximal responses were lowered. Although O₂^.– production by NaCl and KCl required high concentrations associated with hyperosmolarity, the O₂^.– generation induced by NaCl and KCl was significantly greater than that induced simply by hyperosmolarity. Since an NADPH oxidase inhibitor, diphenyleneiodonium chloride, showed a strong inhibitory effect on the trivalent and divalent cation‐induced generation of O₂^.–, it is likely that cation treatments activate the O₂^.–‐generating activity of NADPH oxidase.