The Mechanism of Ion Translocation in Mitochondria. 3. Coupling of K+ Efflux with ATP Synthesis

Rossi, Elisabetta; Gf, Azzone

doi:10.1111/j.1432-1033.1970.tb00853.x

Cited by 81 publications

(25 citation statements)

References 16 publications

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“…The appropriate cation gradients have been shown to reverse both Na+,K+-ATPase activity in the erythrocyte (29) and Ca++-ATPase activity in membrane vesicles of the sarcoplasmic reticulum (30 (32,33) that in valinomycin-treated mitochondria, ATP synthesis may be coupled to the efflux of potassium. As discussed by G'ynn (25), these observations are compatible with the idea that R.uch AT1P synthesis is driven by an electrical potential resulting from potassium efflux mediated by valinomycin.…”

Section: Discussionmentioning

confidence: 99%

A Protonmotive Force Drives ATP Synthesis in Bacteria

Maloney

Kashket

Wilson

1974

Proc. Natl. Acad. Sci. U.S.A.

147

103

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When cells of Streptococcus lactis or Escherichia coli were suspended in-a potassium-free medium, a membrane potential (negative inside) could be artificially generated by the addition of-the potassium ionophore, valinomycin. In 'response to this inward directed protonmotive force, ATP synthesis catalyzed by the mnembrane-bound'ATPase (EC 3.6.1.3) was observed. (Fig. 1A). Thus, the electrochemical potential of protons (the protonmotive force) provides the driving force for ATP synthesis. The alternative (anaerobic) function of the ATPase is required when protons cannot be extruded by the respiratory chain. Under these conditions, the ATPase couples the hydrolysis of ATP to the electrogenic movement of protons out of the cell (Fig. 11B). The protonmotive force generated by ATP hydrolysis is then utilized by energy-dependent reactions such as the "ATP-linked" transhydrogenase, or the active transport of metabolites.Evidence in support of this anaerobic function of the ATPase has been presented by Harold and his collaborators, who have studied the anaerobe S. fecalis (faecium). They showed that glycolyzing cells establish both a pH gradient (interior alkaline) and a membrane potential (interior negative), and that DCCD inhibits the formation of each of these components of the protonmotive force (17)(18)(19) 1% (w/v)

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Section: Discussionmentioning

confidence: 99%

A Protonmotive Force Drives ATP Synthesis in Bacteria

Maloney

Kashket

Wilson

1974

Proc. Natl. Acad. Sci. U.S.A.

147

103

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“…The stimulation of respiration in the steady state which is accompanied by an increased rate of K+ release, is assumed to be due t o an increased leakiness of the membrane (cf. Fig.12 and Discussion of [20] also presented evidence that in steady state conditions the proton motive force, calculated from the distribution of K+ and H+ in the inner and outer mitochondrial spaces, is some 240 mV, this satisfying the thermodynamic requirements for the ATP synthesis. However, the observation that mitochondria are able to produce a very high Kg/Ko ratio does not exclude that oxidative phosphorylation be maintained a t much lower Kt/Ko ratios.…”

Section: Resultsmentioning

confidence: 54%

“…The P/O ratio was calculated from the amount of ADP added and the total oxygen uptake during the ADP stimulated respiration. It is to be noted that addition of ADP to valinomycin treated mitochondria caused an efflux of K+ which was coupled to ATP synthesis [20] this resulting in a P/O ratio higher than 3.0. From an initial value of 5.2 a t 0.45 mM K O the P/O ratio decreased proportionally to the increase of the external K+ concentration.…”

Section: Resultsmentioning

confidence: 99%

The Mechanism of Ion Translocation in Mitochondria. 2. Active Transport and Proton Pump

Massari

1970

Eur J Biochem

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A model is presented for the energy linked accumulation of cations. It is assumed that the H+ carrier is energized a t the inner surface and acquires an increased nucleophilicity. The translocation of the protonated carrier to the outer surface involves a de-energization. Kinetic equations have been developed and have been tested in respect to several parameters of the process of active transport. The effects of the internal and external K+ and H+ concentrations and of valinomycin are in agreement with the predictions of the equations. Also, in agreement with the model, there is competition of divalent cations with the K+ uptake.The steady state respiration of valinomycin treated mitochondria is shown to be very low although there is a large Kf concentration gradient. Swelling of mitochondria causes a considerable increase in the steady state respiration. There is a parallel lowering of the K+ concentration gradient. By measuring the P/O ratio in valinomycin treated mitochondria it is found that phosphorylation is abolished only when the proton motive force, calculated according to Mitchell, becomes lower than 122 mV. The low rate of state 4 respiration is assumed in the present model, where the thermodynamic potential of the internal cations is higher than that of the external ones, t o be a function of the amount of carrier in the energized and ground states.The steps involved in the transmembrane K+ translocation are also analyzed by using inhibitors of the surface binding, such as Li+, and of the translocation of cations in the hydrophobic phase, such as local anesthetics. It is concluded that three steps in series are involved for the cation translocation. These occur a t the surface of the mitochondria, in the hydrophobic phase and a t the carrier level.The energy linked accumulation of cations within the mitochondria has prompted a great number of investigations in recent years (for reviews see [1,2]). Yet very little is known about the molecular mechanism of the process. Basic questions such as the thermodynamic potential of the intramitochondrial ions, the step where energy is invested, have not yet been answered. Two different types of mechanisms have been proposed. According to the chemical hypothesis [3-71 cation carriers are present in the mitochondrial membrane. The carriers are energized by high energy intermediates and operate the accumulation of the cations. The process requires neither a membrane potential nor a special role for protons in ion transport.According to the chemiosmotic hypothesis [8-111 the cations diffuse passively along an electrical gradient which is formed through the primary electrogenic reaction of electron transport or ATP hydrolysis. I n this hypothesis the cations are a t electrochemical equilibrium and the link between metabolism and transport is the membrane potential.I n a preceding paper [12] we have proposed a mechanism for ion translocation which assumes the presence of proton carriers coupling the electroneutral translocation of protons, of univalent and divalent c...

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“…The phosphorylative activity was measured by adding 0.5-2 mg protein of sonic fragments to a standard incubation medium of 2.5 ml containing 40 mM P, pH 7.0, 1 or 10 mM MgCI2 l00pM AMP, 200pM ADP, 0.2 M sucrose, 2.5 mg hexokinase (Sigma type IV), 20 mM glucose and 1 mM NADH or 2 mM succinate. After 10 or 15 min the reaction was blocked with perchloric acid and the system analyzed for the presence of glucose 6-phosphate fluorimetrically [30].…”

Section: Methodsmentioning

confidence: 99%

Anion and Amine Uptake and Uncoupling in Submitochondrial Particles

Azzone

Gutweniger

Viola

et al. 1976

European Journal of Biochemistry

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1. Unlike chloroplasts, submitochondrial particles are not uncoupled by nigericin + KC1 or NH,CI. Also the uncoupling effect of lipophilic anions is largely independent of the addition of weak bases.2. Low concentrations of permeant anions cause a shift of the steady-state energy level rather than a cycle of energy utilization. The degree of inhibition of ATP synthesis by tetraphenylboron is larger than required for the uptake of the anion.3. Lipophilic anions such as bromthymolblue, bromcresolpurple, and 8-anilino-I-napthalene sulphonate cause a pH-independent, 50 uncoupling in submitochondrial particles at concentrations of 3, 30 and 30 pM, respectively. The passive interaction of bromthymolblue and bromcresolpurple appears as a pH-dependent distribution between two phases. ATP causes a pH-independent slight shift in the anion distribution, with negligible anion accumulation.4. Addition of amines to energized submitochondrial particles results in two types of effects : uptake of amines and uncoupling. While in chloroplasts amine uptake and uncoupling are closely associated, this is not the case in submitochondrial particles. The uncoupling effect is observed only with lipophilic and not with hydrophilic amines, and the degree of uncoupling increases with the lipophilicity of the amines. The amine uptake, on the other hand, is accompanied by negligible uncoupling.5. While the uptake of amines is dependent on the presence of non-permeant anions, such as C1-, the uncoupling effect is independent of C1-. Furthermore the amine uncoupling is markedly enhanced by lipophlic anions.6. The view is discussed that the uncoupling effect of lipophilic anions and lipophilic amines in submitochondrial particles is due to a catalytic energy dissipation rather than to a stoichiometry energy utilization. The molecular mechanism of uncoupling presumably involves a cycling of charges after a perturbation of the membrane structure.When active transport takes place other energylinked reactions are inhibited [l]. Although the inhibition is loosely indicated as "uncoupling" it has been realized that the uncoupling due to active transport is different in nature from the classical uncoupling. In fact the former is due to stoichiometric energy utilization for ion transport while the latter is due to catalytic energy dissipation. The former is accompanied by a stoichiometric cycle of energy utilization, denoted as state 4-state 3 -state 4 transition, while the latter is accompanied by a shift of the steady-state energy level. While the distinction between stoichiometric energy utilization, linked to ion uptake, and catalytic energy dissipation, due to damage or uncoupler cycling, is clearly established in intact mitochondria, this is not the case in submitochondrial particles.Skulachev el al.[2] reported an uncoupling effect of lipophilic anions in submitochondrial particles and suggested it be equivalent to the energy utilization accompanying cation uptake in mitochondria. Christiansen et al.

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The Mechanism of Ion Translocation in Mitochondria. 3. Coupling of K+ Efflux with ATP Synthesis

Cited by 81 publications

References 16 publications

A Protonmotive Force Drives ATP Synthesis in Bacteria

A Protonmotive Force Drives ATP Synthesis in Bacteria

The Mechanism of Ion Translocation in Mitochondria. 2. Active Transport and Proton Pump

Anion and Amine Uptake and Uncoupling in Submitochondrial Particles

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