Facilitated transport of di- and trinitrophenolate ions across lipid membranes by valinomycin and nonactin

The conductance of black lipid membranes in the presence of 2,4,6-trinitrophenol (or 2,4-dinitrophenol) is considerably enhanced, if the cation carriers valinomycin, enniatin B or nonactin are added. The effect is, however, largely independent of the cation concentration and is identical for the cations Li⁺, Na⁺ and Ba²⁺. This finding, as well as the sign and magnitude of the diffusion potential in the presence of a gradient of picrate are consistent with the assumption that the transport of picrate anions is facilitated by the above-mentioned macrocyclic compounds, but that cations are not directly involved. A model is suggested which, based on the generation of mobile defect structures by the incorporation of large molecules, allows one to explain facilitated transport without the assumption of stable chemical bonds between a carrier and its transported substrate.If K⁺ is present in the aqueous phase, the conductance is largely determined by the permeation of the cation complexes of valinomycin and nonactin. The conductance is, however, increased by adsorption of picrate anions to the membrane surface. The negative surface potential generated by the adsorption layer seems to be responsible for the saturation of the conductance at high picrate concentrations in the absence of valinomycin and nonactin.     

A circular dichroism study of valinomycin barium ion complex

Complex formation of valinomycin with Ba²⁺ ions was investigated by circular dichroism spectroscopy. The results indicated that Ba²⁺ forms entirely different types of complexes when compared with K⁺. The data with perchlorate salt showed evidence for the formation of less stable V₂C (peptide sandwich), VC (1:1), and VC₂ (ion sandwich) complexes followed by a stable final complex upon gradual addition of salt (V stands for valinomycin and C for the cation). This final complex possibly has a flat structure with no internal hydrogen bonds, similar to that of valinomycin in highly polar solvents. The possible complexation mechanism and the role played by anions and isopropyl side chains are highlighted.     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Stability of sodium and potassium complexes of valinomycin

Stability constants of sodium and potassium complexes of valinomycin in some alcohols and water—organic solvent mixtures have been determined by titration, using circular dichroism to monitor complex formation. Constants range from 10¹ to 10⁶ M⁻¹. Stability of the potassium and sodium complexes increases with decreasing dielectric constant, but the ratio of the constants remains about 10³–10⁴. As others have shown, a similar selectivity for K⁺ is observed in a number of other types of measurements involving valinomycin. These include the permeability and conductance ratios which characterize the selectivity of cation transport through membranes and the ratio of salt extraction equilibrium constants. On the basis of data presented here, and elsewhere, it is suggested that conformational constraints within the depsipeptide part of the complexes aid ion selectivity and that differences in cation solvation and carbonyl ligand binding energies make an important, roughly equal, contribution.     

Cation transport and electrogenesis byStreptococcus faecalis

Summary Uptake of the lipid-soluble cations dibenzyldimethylammonium (DDA⁺) and triphenylmethylphosphonium (TPMP⁺) byStreptococcus faecalis is biphasic. The initial phase is a rapid binding of the ions which does not require a source of metabolic energy and apparently consists of cation exchange at the cell surface. Upon addition of glucose further uptake of the cations occurs, by exchange for Na⁺ and H⁺. Evidence is presented suggesting that this metabolic uptake of DDA⁺ and TPMP⁺ is not due to active transport. It rather appears that uptake results from the generation of an electrical potential, interior negative, by the extrusion of H⁺ and, indirectly, of Na⁺. Accumulated DDA⁺ and TPMP⁺ are discharged by proton-conducting uncouplers. The cationconducting antibiotics valinomycin, monactin, nigericin and monensin do not inhibit uptake. Potassium and, under certain conditions, H⁺ displace DDA⁺ and TPMP⁺. Generation of an electrical difference across the membrane was verified by the accumulation of K⁺ in the presence of valinomycin. The concentration ratios achieved correspond to potentials of the order of –150 to –200 mV.     

Characteristics of thyroxine swelling in skeletal muscle mitochondria: Relationship to valinomycin swelling and swelling in the absence of Mg2+

Summary Divalent cation-depleted skeletal muscle mitochondria undergo energy-dependent swelling in the presence of thyroxine analogues+Mg²⁺, as well as in the presence of valinomycin or the absence of Mg²⁺. ATP-supported swelling shows a K⁺-specificity in the presence of thyroxine analogues or valinomycin, in contrast to a Na⁺-specificity in the absence of Mg²⁺. Substrate-supported swelling shows a K⁺-specificity in the presence of valinomycin but fails to show an alkali metal cation specificity under the other two swelling conditions. All three kinds of swelling show a permeant anion dependency. Although Mg²⁺ inhibits the swelling which occurs in its absence and also inhibits uncoupling of respiration, even in the presence of valinomycin, nevertheless Mg²⁺ does not inhibit the energy-dependent swelling which occurs in the presence of valinomycin or thyroxine analogues. The findings show that thyroxine does not promote swelling simply because it chelates Mg²⁺. Rather, they show that thyroxine promotes a selective change in accessibility of monovalent cations. They suggest that thyroxine in the presence of Mg²⁺ acts at the first coupling site as an electron ccepptor. An observed inhibition of oxygen uptake would appear to be explained on the basis of thyroxine in higher concentration acting as an electron sink. The findings suggest that, as with the lipid-soluble K⁺ carrier, valinomycin, in the presence of Mg²⁺, a change in the status of electrical gradients in the membrane can account for the osmotic swelling observed in the presence of thyroxine analogues.Contribution No. 346 from the Animal Research Institute, Canada Department of Agriculture, Ottawa, Canada.     

Two-phase partition studies of alkali cation complexation by ionophores

Summary The partition of alkali cations and anions between an aqueous and an immiscible organic phase has been studied in the absence and presence of neutral and carboxylic ionophores of the valinomycin and nigericin types, respectively. Cation extraction into the organic phase was augmented considerably by the ionophores, and a cation specificity of K⁺Rb⁺>Cs⁺Na⁺ was found for all the neutral ionophores tested. Evidence is given that the actual values of ion specificity are a function of the solvent polarity, especially for valinomycin where an inversion of the K⁺/Rb⁺ specificity was observed. The ionophores examined have the following rank order of effectiveness for K⁺ extraction into a standard organic phase consisting of 70% toluene-30%n-butanol: valinomycin>18-crown-6trinactin>enniatin Bdinactin>monactin>nonactin. The ion affinity and selectivity data thus obtained have been compared with data previously reported.In a toluene-butanol solvent, extraction of cations in the absence of ionophores occurs as ion pairs. On the other hand, the neutral ionophores extract the cations by the mechanism of complexation, with the lipophilic anions coextracted as free gegenionic species at lower ionophore complex concentrations. When the concentration of extracted cations exceeds 1×10^–4 m, ion pairing between the ionophore complex and the anion occurs, and this tendency increases with increasing concentration and decreasing polarity of the organic phase. Anion pairing with the complexed cations is much less than for the free cations and this effect appears to be due to the larger distance of closest approach of the anion for the complexed cation.     

Energized transport of potassium ions in the absence of valinomycin by cytochrome c oxidase-reconstituted vesicles

Valinomycin-independent energized uptake of K⁺ was observed in cytochrome c oxidase reconstituted proteoliposome. The rate of K⁺ influx was proportoinal to the magnitude of electron flux. The energized uptake of K⁺ was abolished by p-trifluoromethoxycarbonylcyanide phenylhydrazone or by nigericin. Using the safranine fluorescence technique, it was demonstrated that even in the absence of valinomycin, liposomes and proteoliposomes reconstituted with cytochrome c oxidase are able to discriminate between Na⁺ and K⁺ and show a preference for K⁺ in the presence of excess Na⁺.     

The respiration of brain mitochondria and its regulation by monovalent cation transport

The Na⁺ and K⁺ permeability properties of rat brain mitochondria were determined to explain the influences of these cations upon respiration. A new procedure for isolating exceptionally intact mitochondria with minimal contamination by synaptosomes was developed for this purpose.Respiration was uncoupled by Na⁺ and less so by K⁺. Uncoupling was maximal in the presence of EDTA plus P_i and was decreased by Mg²⁺. Maximal uncoupler-stimulated respiration rates were inhibited by Na⁺ but largely unaffected by K⁺. The inhibition by Na⁺ was relatively insensitive to Mg²⁺. Membrane Na⁺ and K⁺ conductances as well as neutral exchanges (Na⁺/H⁺ and K⁺/H⁺ antiport activities) were determined by swelling measurements and correlated with metabolic effects of the cations.Cation conductance, i.e. electrophoretic Na⁺ or K⁺ permeation, was increased by EDTA (Na⁺ > K⁺) and decreased by Mg²⁺. Magnesium preferentially suppressed Na⁺ conductance so as to reverse the cation selectivity (K⁺ > Na⁺). Neutral cation/H⁺ exchange rates (Na⁺ > K⁺) were not influenced by chelator or Mg²⁺.The extent of cation-dependent uncoupling of respiration correlated best with the inner membrane conductance of the ion according to an empirical relationship derived with the model K⁺ conductor valinomycin. The metabolic influences of Na⁺ and K⁺ can be explained in terms of coupled flow of these ions with protons and their effect upon the H⁺ electrochemical gradient although alternative possibilities are discussed. These in vitro studies are compared to previous observations in situ to assess their physiological significance.     

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(d-Val-l-Pro-l-Val-d-Pro)3

The cyclic dodecapeptide PV, cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃, a structural analogue of the ion-carier valinomycin, increase the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na⁺ < NH₄⁺ < K⁺ < Cs⁺ < Rb⁺. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanims). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.     

K+-independent effects of valinomycin in photosynthetic systems

With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K⁺. NH₄Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH₄Cl plus valinomycin but not by either alone. K⁺ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH₄Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg²⁺-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H⁺/e^– ratio. These contrasting activities in chloroplasts are not easily explained.Contribution number 389 of the Charles F. Kettering Research Laboratory.     

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Summary The uptake and binding of the lipophilic cations ethidium⁺, tetraphenylphosphonium⁺ (TPP⁺), triphenylmethylphosphonium⁺ (TPMP⁺), and tetraphenylarsonium⁺ (TPA⁺) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K' _o) and the internal matrix volume and matrix face of the inner membrane (K' _i) were determined and were utilized to estimate the membrane potential from the cation accumulation factorR _c according to the relation =RT/ZF ln [(R _cV_o–K'_o)/(V_i+K'_i)] whereV _o andV _i are the volume of the external medium and the mitochondrial matrix, respectively, andR _c is the ratio of the cation content of the mitochondria and the medium. The values of estimated from this equation are in remarkably good agreement with those estimated from the distribution of⁸⁶Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.     

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⁺ ? Cs⁺ > Rb⁺ > K⁺ Na⁺ > Li⁺ ? 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 nondifussible anions and (2) a selective permeability of the lysosomal membrane to cations.     

Alkali Cation/Sucrose Co-transport in the Root Sink of Sugar Beet

The mechanism of sucrose transport into the vacuole of root parenchyma cells of sugar beet was investigated using discs of intact tissue. Active sucrose uptake was evident only at the tonoplast. Sucrose caused a transient 8.3 millivolts depolarization of the membrane potential, suggesting an ion co-transport mechanism. Sucrose also stimulated net proton efflux. Active (net) uptake of sucrose was strongly affected by factors that influence the alkali cation and proton gradients across biological membranes. Alkali cations (Na⁺ and K⁺) at 95 millimolar activity stimulated active uptake of sucrose 2.1- to 4-fold, whereas membrane-permeating anions inhibited active sucrose uptake. The pH optima for uptake was between 6.5 and 7.0, pH values slightly higher than those of the vacuole. The ionophores valinomycin, gramicidin D, and carbonyl cyanide m-chlorophenylhydrazone at 10 micromolar concentrations strongly inhibited active sucrose uptake. These data are consistent with the hypothesis that an alkali cation influx/proton efflux reaction is coupled to the active uptake of sucrose into the vacuole of parenchyma cells in the root sink of sugar beets.     

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles

The Mg²⁺-dependent, K⁺-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H⁺ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth).Valinomycin stimulated both dye uptake and K⁺-ATPase (valinomycin-stimulated K⁺-ATPase); stimulation by valinomycin was due to increased K⁺ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K⁺-ATPase and H⁺ accumulation as an index, the sequence for anion permeation was NO₃^? > Br^? > Cl^? > I^? > acetate ≈ isethionate. When permeability to both K⁺ and H⁺ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K⁺-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an ‘uncoupling’ of ATP hydrolysis from H⁺ accumulation.Thiocyanate interacts with valinomycin inhibiting the typical action of the K⁺ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN^? to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K⁺-ATPase, it appears that SCN^? is a very     

Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects on electron transport and reconstitution of photophosphorylation

1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C³⁺ > C²⁺ > C⁺.2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor.3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane.4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C³⁺ > C²⁺ > C⁺, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding.5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane.6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations.7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory.     

The effect of valinomycin in fibroblasts from patients with fatty acid oxidation disorders

Disorders of the carnitine cycle and of the beta oxidation spiral impair the ability to obtain energy from fats at time of fasting and stress. This can result in hypoketotic hypoglycemia, cardiomyopathy, cardiac arrhythmia and other chronic medical problems. The in vitro study of fibroblasts from patients with these conditions is impaired by their limited oxidative capacity. Here we evaluate the capacity of valinomycin, a potassium ionophore that increases mitochondrial respiration, to increase the oxidation of fatty acids in cells from patients with inherited fatty acid oxidation defects. The addition of valinomycin to fibroblasts decreased the accumulation of the lipophilic cation tetraphenylphosphonium (TPP⁺) at low concentrations due to the dissipation of the mitochondrial membrane potential. At higher doses, valinomycin increased TPP⁺ accumulation due to the increased potassium permeability of the plasma membrane and subsequent cellular hyperpolarization. The incubation of normal fibroblasts with valinomycin increased [¹⁴C]-palmitate oxidation (measured as [¹⁴C]O₂ release) in a dose-dependent manner. By contrast, valinomycin failed to increase palmitate oxidation in fibroblasts from patients with very long chain acyl CoA dehydrogenase (VLCAD) deficiency. This was not observed in fibroblasts from patients heterozygous for this condition. These results indicate that valinomycin can increase fatty acid oxidation in normal fibroblasts and could be useful to differentiate heterozygotes from patients affected with VLCAD deficiency.     

Discrimination between Alkali Metal Cations by Yeast : II. Cation interactions in transport

K⁺ is a competitive inhibitor of the uptake of the other alkali metal cations by yeast. Rb⁺ is a competitive inhibitor of K⁺ uptake, but Li⁺, Na⁺, and Cs⁺ act like H⁺. At relatively low concentrations they behave as apparent noncompetitive inhibitors of K⁺ transport, but the inhibition is incomplete. At higher concentrations they inhibit the remaining K⁺ transport competitively. Ca⁺⁺ and Mg⁺⁺ in relatively low concentrations partially inhibit K⁺ transport in an apparently noncompetitive manner although their affinity for the transport site is very low. In each case, in concentrations that produce "noncompetitive" inhibition, very little of the inhibiting cation is transported into the cell. Competitive inhibition is accompanied by appreciable uptake of the inhibiting cation. The apparently noncompetitive effect of other cations is reversed by K⁺ concentrations much higher than those necessary to essentially "saturate" the transport system. A model is proposed which can account for the inhibition kinetics. This model is based on two cation-binding sites for which cations compete, a carrier or transporting site, and a second nontransporting (modifier) site with a different array of affinities for cations. The association of certain cations with the modifier site leads to a reduction in the turnover of the carrier, the degree of reduction depending on the cation bound to the modifier site and on the cation being transported.     

Sodium and potassium binding by rat liver cell microsomes

The effects of ion concentration, pH, and presence of competing ions on the sodium and potassium binding properties of rat liver cell microsomes were studied. Typical adsorption isotherms were obtained in the concentration dependence studies, with saturation being reached when 1.2 to 1.4 m.eq. cations were retained per gm. of microsome Kjeldahl nitrogen. The retention was shown to be due to a binding to specific sites rather than to a trapping of the cations. The binding showed a sharp pH dependence in the range 6.0 to 7.5. The presence of one cation depressed the binding of the other, indicating that Na⁺ and K⁺ as well as H⁺ ions compete for the same sites. Potassium was bound slightly more strongly than sodium, while hydrogen was bound about 10⁵ times more strongly than either. Calculations show that the binding follows the simple mass law. Similarities between adsorption by microsomes and adsorption by synthetic cation exchange resins are discussed and compared to some of the characteristics of electrolyte behavior in living systems. A possible ion exchange elution, active cation transport mechanism is suggested, involving the preferential elution of Na⁺ out of the cell by H⁺ ions produced by metabolism.     

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).