Mechanism of active shrinkage in mitochondria. II. Coupling between strong electrolyte fluxes.

1. Addition of succinate to valinomycin-treated mitochondria incubated in KCL causes a large electrolyte penetration. The process depends on a steady supply of energy and involves a continuous net extrusion of protons. Rates of respiration and of electrolyte penetration proceed in a parallel manner. 2. A passive penetration of K+ salt of permeant anions occurs in respiratory-inhibited mitochondria after addition of valinomycin. Addition of succinate at the end of the passive swelling starts an active extrusion of anions and cations with restoration of the initial volume. The shrinkage is accompanied by a slow reuptake of protons. The initiation of the active shrinkage correlates with the degree of stretching of the inner membrane. The extrusion of electrolytes is inhibited by nigericin, while it is only slightly sensitive to variations of the valinomycin concentration larger than two orders of magnitude. 3. Passive swelling and active shrinkage occurs also when K+ is replaced by a large variety of organic cations. The rate of organic cation penetration is enhanced by tetraphenylboron, while the rate of electrolyte extrusion is insensitive to variation of the tetraphenylboron concentration. 4. Active shrinkage, either with K+ or organic cation salts, is inhibited by weak acids. The phosphate inhibition is removed by SH inhibitors. The active shrinkage is also inhibited by mersalyl to an extent of about 60%. 5. Three models of active shrinkage are discussed: (a) mechanoprotein, (b) electrogenic proton pump, and (c) proton-driven cation anion pump.     

Mitochondrial oscillation and activation of H+/cation exchange

Mitochondria incubated aerobically in the presence of tetrapropylammonium and weak acids and in the presence of trace amounts of tetraphenylboron undergo a series of damped oscillations reflecting cycles of osmotic swelling and shrinkage. The matrix volume changes are consequent to transport of tetrapropylammonium catalytically stimulated by tetraphenylboron. The amplitude and frequency of the oscillations increase with the concentration of tetrapropylammonium, as required for critical rates and extents of ion influx. Addition of bovine serum albumin abolishes both the uptake of tetrapropylammonium and the oscillations. Volume oscillations are paralleled by cyclic activation and depression of the respiratory rate. Two lines of evidence suggest that the train of damped oscillations depends on the cyclic activation of an electroneutral exchange of H⁺ with organic cations rather than on cyclic uncoupling. First, further increase of cation permeability due to a pulse of tetraphenylboron, after initiation of cation efflux, restores cation influx. Second, addition of Mg²⁺, which abolishes the oscillations, has a much more marked inhibitory effect on the process of cation efflux than on cation influx. Conversely, addition of A23187, which removes membrane-bound Mg²⁺, promotes cation efflux and thus the oscillations. It is suggested that, in the present system, stretching of the inner membrane and Mg²⁺ depletion result in activation of an electroneutral H⁺/organic cation exchange, and that cyclic activation of this reaction results in damped oscillations.     

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.     

Tributylbenzylammonium (TBBA+)-dependent fusicoccin (FC)-induced H+ extrusion in maize roots: relationship between the stimulating effects of TBBA+ on H+ extrusion and on Cl- efflux

The mechanism of the stimulating effect of lipophilic cations on H⁺ extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [³H]tributylbenzylammonium ([³H]TBBA⁺), the most active lipophilic cation on H⁺ extrusion, indicated that although a relevant fraction of TBBA⁺ taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA⁺ uptake and that of H⁺ extrusion. On the other hand, the lipophilic cations active on H⁺ extrusion (TBBA⁺ and dibenzyldimethylammonium (DDA⁺)), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl^-, while tetramethylammonium (TMA⁺), inactive on H⁺ extrusion, did not. The stimulation of Cl^- efflux by TBBA⁺ was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA⁺-induced H⁺ extrusion. These results suggest that the stimulation of H⁺ extrusion by TBBA⁺ depends on its effect on Cl^- efflux rather than on its penetration into the cells.Abbreviations DDA⁺ dibenzyldimethylammonium - FC fusicoccin - 3-O-MG 3-O-methyl glucose - PD transmembrane electric potential difference - TBBA⁺ tributylbenzylammonium - TCF tissue concentration factor - TMA⁺ tetramethylammonium - TPB^- tetraphenylboron     

Mechanism of active shrinkage in mitochondria I. Coupling between weak electrolyte fluxes

1. A passive penetration of (NH₄)₂ HPO₄ or of K₂HPO₄+nigericin occurs in respiratory-inhibited liver mitochondria. Addition of succinate at the end of the passive swelling initiates a shrinkage phase which leads to restoration of the initial mitochondrial volume. The rate and time of onset of the active shrinkage depend on the degree of stretching of the mitochondrial membrane. The rate of active shrinkage increases proportionally to the concentration of nigericin while it is strongly inhibited by valinomycin.2. A number of SH inhibitors such as N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuriphenylsulphonate, dithiobisnitrobenzoate, exert a marked enhancing effect on the rate of shrinkage. The enhancing effect parallels titration of the phosphate carrier and inhibition of the passive phosphate influx. The above SH inhibitors do not inhibit passive phosphate efflux. In contrast, mersalyl is a powerful inhibitor of the rate of active shrinkage. The inhibition parallels that on phosphate passive efflux and requires higher mersalyl concentrations in respect to inhibition of phosphate influx.3. The active shrinkage is discussed in terms of (a) a mechanoenzyme, (b) an electrogenic proton pump and (c) a proton-driven P_i pump.     

On the mechanism of action of polyether XXVIII at site I of the electron-transfer chain in rat liver mitochondria

In rat liver mitochondria, the macrocyclic polyether, dibenzo-18-crown-6 (polyether XXVIII) inhibits the oxidation of NAD-dependent substrates, as stimulated by ADP, uncouplers and valinomycin plus K⁺. It does not inhibit the oxidation of succinate. It is concluded that polyether XXVIII inhibits electron transfer in the NADH-CoQ span of the respiratory chain. This is a process that is reversed by menadione. Inhibition of oxidation of NAD-dependent substrates in K⁺-depleted mitochondria induced by the polyether is reversed by concentrations of K⁺ higher than 60 mM, and also by Li⁺, a cation that does not complex with polyether XXVIII. As assayed by swelling mitochondria, reversal of the inhibition of electron transfer is accompanied by influx of monovalent cations. Polyether XXVIII also inhibits in submitochondrial particles the aerobic oxidation of NADH, but not that of succinate; this inhibition is also reversed by K⁺ at high concentrations, and Li⁺. The data are consistent with the hypothesis that a monovalent cation is required for maximal rates of electron transport in the NADH-CoQ span of the respiratory chain.     

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.     

THE PENETRATION OF THE MEMBRANE OF BRAIN MITOCHONDRIA BY ANIONS

The permeability of the membrane of rat brain non-synaptosomal mitochondria, towards inorganic and substrate anions, was assessed by measuring the rate of swelling that occurred when mitochondria were suspended in an iso-osmotic solution of a permeant anion, in the presence of a permeant cation such as NH⁺₄ or K⁺ in the presence or absence of valinomycin. In NH⁺₄-phosphate swelling was higher than it was in KCI or K⁺-phosphate, which showed the prevalence of the mechanism of phosphate transport previously demonstrated in liver mitochondria. The entry of succinate and L-malate seemed to require the presence in the inner mitochondrial membrane of specific carriers. as previously postulated for liver mitochondria, but the rate of swelling of brain mitochondria was lower than that of liver organelles. In K⁺-succinate, in the presence of antimycin, added ATP induced swelling and this was attributable to the simultaneous permeation both of the anion and the cation. Fumarate did not penetrate into brain mitochondria. Practically no swelling was recorded in NH⁺₄ or K⁺-citrate, which indicated that this anion penetrated poorly into the isolated brain mitochondria even in the presence of malate. Swelling occurred in NH⁺₄-L-glutamate in the presence of rotenone, and the entry of this anion seemed to follow a gradient of concentration although the presence of a specific translocator in the inner mitochondrial membrane might be concerned. The entry of glutamate was independent of that of phosphate and N-ethylmaleimide appeared to be a specific inhibitor of this entry. Swelling in K⁺-L-glutamate, in the presence of rotenone, was enhanced by the addition of valinomycin or ATP but in the latter case when osmotic equilibrium was reached swelling was not reversed by oligomycin. In conclusion, the lesser extent of swelling of isolated brain mitochondria compared with liver mitochondria could be attributed to the heterogeneity of the populations of these organelles, each population possessing its own characteristics of membrane permeability. Observations of electron micrographs of brain mitochondria incubated in iso-osmotic substrate anions confirmed the heterogeneous rate of swelling of these particles.     

The use of potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Spectrophotometric measurements

Renal transport of four different categories of organic solutes, namely sugars, neutral amino acids, monocarboxylic acids and dicarboxylic acids, was studied by using the potential-sensitive dye 3,3′-diethyloxadicarbocyanine iodide in purified luminal-membrane and basolateral-membrane vesicles isolated from rabbit kidney cortex. Valinomycin-induced K⁺ diffusion potentials resulted in concomitant changes in dye–membrane-vesicle absorption spectra. Linear relationships were obtained between these changes and depolarization and hyperpolarization of the vesicles. Addition of d-glucose, l-phenylalanine, succinate or l-lactate to luminal-membrane vesicles, in the presence of an extravesicular>intravesicular Na⁺ gradient, resulted in rapid transient depolarization. With basolateral-membrane vesicles no electrogenic transport of d-glucose or l-phenylalanine was observed. Spectrophotometric competition studies revealed that d-galactose is electrogenically taken up by the same transport system as that for d-glucose, whereas l-phenylalanine, succinate and l-lactate are transported by different systems in luminal-membrane vesicles. The absorbance changes associated with simultaneous addition of d-glucose and l-phenylalanine were additive. The uptake of these solutes was influenced by the presence of Na⁺-salt anions of different permeabilities in the order: Cl⁻>SO₄²⁻>gluconate. Addition of valinomycin to K⁺-loaded vesicles enhanced uptake of d-glucose and l-phenylalanine in the presence of an extravesicular>intravesicular Na⁺ gradient. Gramicidin or valinomycin plus nigericin diminished/abolished electrogenic solute uptake by Na⁺- or Na⁺+K⁺-loaded vesicles respectively. These results strongly support the presence of Na⁺-dependent renal electrogenic transport of d-glucose, l-phenylalanine, succinate and l-lactate in luminal-membrane vesicles.     

Effect of valinomycin on ion transport in bacterial cells and on bacterial growth

The antimicrobial action of valinomycin relative to the K⁺ and Na⁺ contents of the medium has been investigated in several species of bacteria, particularly in Streptococcus faecalis, which effects energy-linked transport exclusively via degradation of glycolytic ATP, Micrococcus lysodeikticus, effecting active ion transport by respiration and Staphylococcus aureus, the energy-dependent ion transport of which is due to both glycolytic ATP degradation and respiration. It was demonstrated that valinomycin does not act on K⁺ transport in the glycolysing cells in the same manner as it does on respiring cells under similar conditions. Addition of valinomycin to respiring cells leads to an increase in K⁺ influx against the concentrational gradient in both growing and resting cells. In contrast to this, antibiotic-treated glycolysing cells experience passive K⁺ outflow down the concentrational gradient. It was thus concluded that the electrical potential cannot be the driving force for the energy-linked K⁺ transport in glycolysing cells.     

Respiration-dependent contraction of swollen heart mitochondria: Participation of the K+/H+ antiporter

Respiration-dependent contraction of heart mitochondria swollen passively in K⁺ nitrate is activated by the ionophore A23187 and inhibited by Mg²⁺. Ion extrusion and osmotic contraction under these conditions are strongly inhibited by quinine, a known inhibitor of the mitochondrial K⁺/H⁺ antiporter, as measured in other systems. The inhibition by quinine is relieved by the exogenous antiporter nigericin. Respiration-dependent contraction is also inhibited by dicyclohexylcarbodiimide (DCCD) when reacted under conditions known to inhibit K⁺/H⁺ antiport (Martinet al., J. Biol. Chem. 259, 2062–2065, 1984). These studies strongly support the concept that K⁺ is extruded from the matrix by the endogenous K⁺/H⁺ antiporter and that inhibition of this component by quinine or DCCD inhibits respiration-dependent contraction. The extrusion of K⁺ nitrate is accompanied by a respiration-dependent efflux of a considerable portion of the endogenous Mg²⁺. This Mg²⁺ efflux does not occur in the presence of nigericin or when the mitochondrial Na⁺/H⁺ antiporter is active. Mg²⁺ efflux may take place on the K⁺/H⁺ antiporter. DCCD, reacted under conditions that do not result in inhibition of the K⁺/H⁺ antiporter, blocks a monovalent cation uniport pathway. This uniport contributes to futile cation cycling at elevated pH, and its inhibition by DCCD stimulates respiration-dependent contraction.     

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles.

Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na⁺ extrusion through an antiport system, and the ability of K⁺ and Cl^? to act as counterions to the electrogenic movement of H⁺. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K⁺. The vesicle membrane is poorly permeable to K⁺, as shown by: lack of a K⁺ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K⁺, and direct measurements of the slow rate of K⁺ influx during illumination. The finding that the rate of K⁺ uptake is a linear function of external K⁺ concentration between 0 and 1 m is inconsistent with the existence of a specific K⁺ permeation mechanism in these vesicles. Since at external K⁺ concentrations < 1.4 m the extrusion of Na⁺ during illumination proceeds much more rapidly than K⁺ influx, it must be concluded that the vesicles also lose Cl^? and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K⁺ is increased, either by increasing the external K⁺ concentration or by adding valinomycin.     

Cation regulation in Anacystis nidulans

Summary Anacystis nidulans accumulates K⁺ in preference to Na⁺. The majority of the internal K⁺ exchanges with ⁴²K by a first order process at rates of about 1.3 pequiv·cm^-2·sec^-1 in the light and 0.26 pequiv·cm^-2·sec^-1 in the dark. Although the K⁺/K⁺ exchange was stimulated by light and inhibited by 10^-4 M CCCP and 10^-5 M DCMU there are several indications that this cation is passively distributed in Anacystis. Inhibition of the exchange by CCCP and DCMU occurred at concentrations greater than those required to inhibit photosynthesis and the K⁺ fluxes were stimulated by low temperatures. Moreover, although valinomycin stimulated the exchange this compound did not induce a net K⁺ leak. Assuming K⁺ is passively distributed and in free solution within the cytoplasm, as indicated by osmotic studies, would imply that there is an active Na⁺ extrusion pump operating in this organism. As yet there are no firm conclusions about the nature of the energy source for this efflux pump.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Dissociation of H + extrusion from K+ uptake by means of lipophilic cations

Abstract Dissociation of active H⁺ extrusion (?ΔH⁺) from K⁺ uptake in pea and maize root segments was attempted by substituting K⁺ in the incubation medium with lipophilic cations assumed to enter the cell by passive, non-specific, permeation through the lipid component of the plasmalemma. Among the compounds tested, tributylbenzylammonium significantly stimulated ?ΔH⁺ in the absence of other monovalent cations in the medium. This effect was much more evident when the experiment was carried out in the presence of fusicoccin, which strongly stimulates proton extrusion and monovalent cation uptake, and hyperpolarizes the trans-membrane electric potential in these materials. Also the lipophilic cations tetraphenylphosphonium, dimethyldibenzylammonium and hexylguanidine markedly stimulated FC-promoted ?ΔH⁺. Octylguanidine at a low concentration induced an early stimulation followed by a strong inhibition of ?ΔH⁺. A complete lack of additivity was observed between the effects of lipophilic cations and that of K⁺ on H⁺ extrusion. Lipophilic cations severely inhibited K⁺ uptake. These data are interpreted as supporting the view of an electric, rather than a chemical, (namely, involving the same carrier system) nature of the coupling of active H⁺ extrusion with K⁺ influx.     

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.     

Active ion uptake and maintenance of cation-anion balance: A critical examination of their role in regulating rhizosphere pH

The processes responsible for maintenance of cation-anion balance in plants and their relation to active ion accumulation and changes in rhizosphere pH are outlined and discussed. The major processes involved are: (1) accumulation and degradation of organic acids which occur in the plant mainly as organic acid anions (and their transfer within the plant) and (2) extrusion of H⁺ or OH^– into the rhizosphere. The relative importance of the two processes is determined by the size of the excess anion or cation uptake. Indeed, plants typically absorb unequal quantities of nutritive cations (NH₄ ⁺+Ca²⁺+ Mg²⁺+K⁺+Na⁺) and anions (NO₃ ^–+Cl^–+SO₄ ^2–+H₂PO₄ ^–) and charge balance is maintained by excretion of an amount of H⁺ or OH^– which is stoichiometrically equal to the respective excess cation or anion uptake. The mechanisms and processes by which H⁺ and in particular OH^– ions are excreted in response to unequal cation-anion uptake are, however, poorly understood.The contemporary view is that primary active extrusion of H⁺, catalyzed by a membrane-located ATPase, is the major driving force for secondary transport of cations and anions across the plasma membrane. However, the fact that net OH^– extrusion often occurs (since excess anion absorption commonly takes place) implies there is a yet-to-be characterized OH^– ion efflux mechanism at the plasma membrane that is associated with anion uptake. There is, therefore, a need for future studies of the uptake mechanisms and stoichiometry of anion uptake; particularly that of NO₃ ^– which is often the predominant anion absorbed. Another related phenonenon which requires detailed study in terms of cation-anion balance is localized rhizosphere acidification which can occur in response to deficiencies of Fe and P.     

Structure and active transport in the plasma membrane of the tubules of frog kidney

The active transport of organic anions through the plasma membrane of the proximal tubules of frog kidney was studied. For this purpose a marker anion, fluorescein, was used, its flow into the tubules registered by the increase of fluorescense. The kinetics of transport was measured as function of time, concentration of substrate, concentration of a competing acid (p-aminohippuric acid) and temperature. The process is inhibited by strophantin, a specific poison for (Na⁺ + K⁺)-dependent ATPase. These data show that fluorescein transport is effected with the participation of a charged carrier, probably by the downfield mechanism postulated by Mitchell. To confirm this mechanism, a passive flow of K⁺ was created inwards across the membrane of the proximal tubules by means of valinomycin. It led to the discharge of the membrane and to the inhibition of fluorescein transport. Anions are transported downfield across the membrane, probably in a state of complexes with two Na⁺ ions.A magnetic field of 10 000–28 000 oersted inhibits the fluorescein transport strongly. This can be regarded as a proof of the liquid-crystalline structure of biological membranes and demonstrates the importance of this structure for active transport.     

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.     

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.     

Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied.Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux.The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K⁺ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the Δμ_H component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K⁺ and Na⁺.