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 the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts. Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller. It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one. Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane surface in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts.

Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller.

It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one.

Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

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.     

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.     

Effect of membrane surface charge on nickel uptake by purified mung bean root protoplasts

The influence of membrane surface charge on cation uptake was investigated in protoplasts prepared from roots of mung bean (Vigna radiata L.). Confocal laser scanning microscopy showed that a fluorescent trivalent cation accumulated to very high concentrations at the surface of the protoplasts when they were incubated in medium containing low concentrations of Ca or other cations, but that this accumulation could be completely reversed by suppression of membrane surface negativity by high cation concentrations. Influx of 63Ni was strongly reduced by a range of divalent cations. Increasing the Ca concentration in the medium from 25 microM to 10 mM inhibited 63Ni influx by more than 85%. 63Ni influx was also inhibited by 85% by reducing the pH from 7 to 4. Computation of the activity of Ni at the membrane surface under the various treatment conditions showed that Ni uptake was closely correlated with its activity at the membrane surface but not with its concentration in the bulk medium. It was concluded that the effects on Ni uptake of addition of monovalent, divalent and trivalent cations, and of variations in pH are all consistent with the proposition that the activity of Ni at the membrane surface is the major determinant of the rate of Ni influx into mung bean protoplasts. It is proposed that the surface charge on the plasma membrane will influence the membrane transport of most charged molecules into cells.     

Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I-, Fe(CN)6(4-), W(CN)8(4-) are poor reducing agents for Z.+ whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors. A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z.+. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner surface.     

Kinetics of ion translocation across charged membranes mediated by a two-site transport mechanism. Effects of polyvalent cations upon rubidium uptake into yeast cells.

(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5.     

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.     

Interfacial pH modulation of membrane protein function in vivo. Effect of anionic phospholipids.

In yeast cells, the magnitude of the membrane surface potential (phi) is determined to a large extent by the relative amount of anionic phospholipids (Cerbón and Calderón (1990) Biochim. Biophys. Acta 1028, 261-267). When a significant surface potential exists, the pH at the membrane surface (interfacial pH) will be different to that in the bulk suspending medium. We now report that: (1) In cells with higher phi (phosphatidylinositol-rich cells (PI-rich) and phosphatidylserine-rich cells (PS-rich) a 10-times lower proton concentration in the bulk was enough to achieve the maximum transport activity of H(+)-linked transport systems when compared to normal cells. (2) When the phi was reduced by increasing the concentration of cations in the medium, more protons were required to achieve maximum transport, that is, the pH activity curves shifted downwards to a more acidic pH. (3) The magnitude of the downward pH shift was around 2.5-times higher for the more charged membranes. (4) Around 10-times more KCl than MgCl2 was necessary to give an equivalent pH shift, in agreement with their capacity to reduce the phi of artificial bilayers. The interfacial pH calculated from the values of phi indicates that it was 0.4 pH units lower in the anionic phospholipid rich cells as compared to normal cells. The results indicate that membrane surface potential may explain the complex relationship between pH, ionic strength and membrane protein function. Maximum transport activities were found for glutamate at interfacial pH of 4.2-4.8 and were inhibited at interfacial pH = 3.2-3.4, suggesting that surface groups of the carrier proteins with pK values in the region 3.8-4.2 (aspartyl and glutamyl) are involved in binding and/or release of charged substrates.     

Studies on the quaternary structure of class I major histocompatibility complex antigens. Effect of different agents on the interaction between subunits

The effect of temperature, urea, guanidine HCl, ionic and nonionic detergents, organic solvents, chaotropic salts, pH, and divalent cations has been investigated on purified human histocompatibility antigens solubilized by papain (HLApap) or solubilized by sodium cholate (HLAchol). HLApap and HLAchol are fairly stable proteins to agents acting predominantly on hydrogen bonds (temperature, urea) or hydrophobic forces (ionic and nonionic detergents). However, agents which affect ionic interactions (pH, salts, divalent cations) dissociate the molecules into subunits. A single binding site for beta 2-microglobulin with an affinity constant of 1.0 X 10(7) M-1 was found for the alpha chain of HLAchol. The dissociated subunits can be separated by affinity chromatography on Sepharose-rabbit IgG anti-human beta 2-microglobulin and reassociate in vitro when incubated under the appropriate conditions. The results point toward an important role of ionic interactions between subunits in the stabilization of the quaternary structure of HLA.     

The protein-mediated transfer of phosphatidylcholine between membranes. The effect of membrane lipid composition and ionic composition of the medium.

The phosphatidylcholine exchange protein from bovine liver catalyzes the transfer of phosphatidylcholine between rat liver mitochondria and sonicated liposomes. The effect of changes in the liposomal lipid composition and ionic composition of the medium on the transfer have been determined. In addition, it has been determined how these changes affected the electrophoretic mobility i.e. the surface charge of the membrane particles involved. Transfer was inhibited by the incorporation of negatively charged phosphatidic acid, phosphatidylserine, phosphatidylglycerol and phosphatidylinositol into the phosphatidylcholine-containing vesicles; zwitterionic phosphatidyl-ethanolamine had much less of an inhibitory effect while positively charged stearylamine stimulated. The cation Mg2+ and, to a lesser extent, K+ overcame the inhibitory effect exerted by phosphatidic acid, in that concentration range where these ions neutralized the negative surface charge most effectively. Under conditions where Mg2+ and K+ affected the membrane surface charge relatively little inhibition was observed. In measuring the protein-mediated transfer between a monolayer and vesicles consisting of only phosphatidylcholine, cations inhibited the transfer in the order La3+ greater than Mg2+ larger than or equal to Ca2+ greater than K+ = Na+. Inhibition was not related to the ionic strength, and very likely reflects an interference of these cations with an electrostatic interaction between the exchange protein and the polar head group of phosphatidylcholine.     

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.     

Electrostatic Interaction between Plastocyanin and P700 in the Electron Transfer Reaction of Photosystem I-Enriched Particles

The nature of the electron transfer reaction between reducedplastocyanin and P700 oxidized by flash illumination was studiedin P700-enriched Triton subchloroplast fraction 1 particles.An addition of monovalent salts to the suspension at neutralpH increased the reaction rate at low concentrations (>20mM). Salts of divalent cations showed a similar effect at muchlower concentrations (>2 mM), This effect was not dependenton the concentration and the valence of anions. The increaseof rate at low salt concentrations was observed at pH's above5, but below pH 5 the rate was decreased by adding salts. Atabout pH 5, the rate was not affected by salts. Apart from thesesalt effects, the optimum pH for the reaction rate was observedbetween 5.5 and 6.5. The reduction rate depended sigmoidally on the added plastocyaninconcentration at pH 6.8 and 4. A Michaelis-Menten type relationshipwas observed at about pH 5. The half-saturation concentrationof plastocyanin became lower as the salt concentration increasedat pH 6.8, while it became higher by adding salt at pH 4. The effects of salts on the rate of electron donation from othermetalloproteins and artificial electron donors to P700 werealso studied. It is concluded, from the analysis with the Gouy-Chapmantheory, that the net charges on the electron donors and themembrane surfaces mainly determine the response of the P700reduction rate to salt addition. The salt addition changes mainlythe local concentration or accessibility of electron donorsto P700. (Received January 12, 1981; Accepted March 6, 1981)     

Interaction of the nuclear proteins protamine and histone with charged bilayer membranes]

The interaction of nuclear proteins of protamine and histone with neutral and charged BLM was studied. Anion and cation detergents were used to create the surface charge. The surface density of charges in BLM was comparable with that in biomembranes. Protamine and histone increased the electroconductivity of negatively charged BLM for anions and cations correspondingly. It is suggested that the surface charge of the membrane may influence the ion transport directly and indirectly due to the interaction of the membrane structures with charged proteins present in the surrounding medium.     

Membrane surface potential and the reactivity of the System II primary electron acceptor to charged electron carriers in the medium

A hypothesis is proposed to explain the change in the apparent rate constant for the reaction between the primary electron acceptor of System II situated in the thylakoid membrane and the artificial electron acceptors added in the medium. Dark oxidation rate of the primary acceptor by artificial electron acceptors was monitored by measuring the induction of chlorophyll fluorescence in the presence of an electron transport inhibitor, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, in spinach chloroplasts. The apparent rate constant for the oxidation changed widely when the medium pH or salt concentrations were varied, or ionic detergents were added. The change was quantitatively ascribed (1) to the change in the local concentration of electron acceptors at the thylakoid surface due to the electrical potential difference between the surface and the bulk aqueous phase (Gouy-Chapman diffuse double layer theory) and (2) to the situation whereby the apparent rate constant is determined with respect to concentration in the bulk phase.Values for the surface potential in the vicinity of System II were estimated from the change in the apparent rate constant under various conditions. The results closely agreed with those obtained previously from the rate constant of the dark step of the System II-dependent Hill reaction with ferricyanide (Itoh, S. (1978) Plant Cell Physiol. 19, 149–166).Application of the hypothesis to various reactions between the added ionic reagents and the endogenous components in the membrane or between the endogenous components situated in different parts of the membrane is discussed.     

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.     

Transport of K+ and other cations across phospholipid membranes by nonesterified fatty acids

The rate of change of internal pH and transmembrane potential has been monitored in liposomes following the external addition of various cation salts. Oleic acid increases the transmembrane movement of H⁺ following the imposition of a K⁺ gradient. An initial fast change in internal pH is seen followed by a slower rate of alkalinization. High concentrations of the fatty acid enhance the rate comparable to that seen in the presence of nigericin in contrast to the effect of FCCP (carbonyl cyanide p-(tri-fluoromethoxy)phenyl hydrazone) which saturates at an intermediate value. The ability of nonesterified fatty acids to catalyze the movement of cations across the liposome membrane increases with the degree of unsaturation and decreases with increasing chain length. Li and Na salts cause a similar initial fast pH change but have less effect on the subsequent slower rate. Similarly, the main effect of divalent cation salts is on the initial fast change. The membrane potential can enhance or inhibit cation transport depending on its polarity with respect to the cation gradient. It is concluded that nonesterified fatty acids have the capability to complex with, and transport, a variety of cations across phospholipid bilayers. However, they do not act simply as proton/cation exchangers analogous to nigericin nor as protonophores analogous to FCCP. The full cycle of ionophoric action involves a combination of both functions.The authors would like to thank P. Nicholts (Brock University, Canada) for helpful discussions. M.A.S. received a Science and Engineering Research Council studentship and C.E.C. acknowledges the award of a King's College London fellowship followed by a Medical Research Council Training Fellowship.     

Photosystem II oxidation of charged electron donors. Surface charge effects

Reactions occurring on the oxidizing side of Photosystem II have been studied in Tris-washed chloroplasts by monitoring the decay kinetics of EPR signal IIf, arising from the photoinduced oxidation of Z, an intermediate in the electron transport chain between P-680 and the water-splitting enzyme. Upon addition of electron donors, signal IIf follows pseudo-first order decay kinetics with rates dependent on the chemical nature of the donor. Negatively charged donors (I⁻, Fe(CN)⁴⁻₆, W(CN)⁴⁻₈) are poor reducing agents for Z⁺· whereas neutral donors (benzidine, hydroquinone, diphenylcarbazide) are more efficient, their effectiveness paralleling their lipophilicity. The slow signal IIf reduction observed with the charged donors is consistent with the non-polar nature of the thylakoid membrane and a location for Z toward the inner membrane surface. It most probably exists in a hydrophobic site as indicated by the positive correlation between rate constant and lipophilicity for the neutral donors.

A detailed study of the mechanism of Photosystem II reduction by ascorbic acid has been carried out. The pH dependence of the decay kinetics of signal IIf in the presence of this donor is consistent with a model in which both the neutral acid and the ascorbate mono-anion serve as reducing agents to Z⁺·. The second-order rate constant for reduction by the mono-anion is less than that of the neutral acid and is found to vary with the suspension pH. This observation is interpreted to indicate the occurrence of negative charge on the inner membrane surface in the vicinity of Z. Additional experiments, which assessed the effect of mono- and divalent cations and of cationic detergents on the signal IIf reaction rate constants, support both the presence of negative surface charge and its location on the membrane inner 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.