Stefano Massari

Proton electrochemical gradient and rate of controlled respiration in mitochondria.

The correlation between deltamuH, the proton electrochemical potential difference, and the rate of controlled respiration is analyzed. deltamuH (the proton concentration gradient) is measured on the distribution of [3H]acetate, and deltapsi (the membrane potential) on the distribution of 86Rb+, 45Ca2+ and [3H]triphenylmethylphosphonium used either alone or simultaneously. The effects of the addition of ADP + hexokinase (state-3 ADP) and of carbonylcyanide trifluoromethoxyphenylhydrazone (state-3 uncoupler) on respiration and deltamuH are not equivalent: the uncoupler depresses deltamuH more than ADP at equivalent respiratory rates. The effects of the additions of nigericin-valinomycin and of ionophore A23187 (state-3 cation transport) and of carbonylcyanide trifluoromethoxy-phenylhydrazone (state 3-uncoupler) on respiration and deltamuH are also not equivalent: the uncoupler depresses deltamuH more than A23187 and nigericin + valinomycin at equivalent respiratory rate. A23187 is very efficient in stimulating respiration with negligible deltamuH changes.

Proton electrochemical gradient and phosphate potential in mitochondria

The paper reports an analysis of the relationship between deltamuH the proton electrochemical potential difference, and deltaGp, the phosphate potential. Depression of deltamuH and deltaGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b) nigericin (+ valinomycin); (c) KCl (+ valinomycin); and (d) rotenone. The uncoupler depresses deltamuH more than nigericin (+ valinomycin), KCl (+ valinomycin) and rotenone at equivalent deltaGp. The deltaGp/deltamuH ratio is about 3 at high values of deltamuH. When deltaGp and deltamuH are depressed by nigericin (4 valinomycin) the deltaGp/deltamuH ratio remains constant. When deltaGp and deltamuH are depressed by uncouplers, the deltaGp/deltamuH ratio increases hyperbolically tending to infinity while deltamuH tends to zero. The absence of constant proportionality between deltaGp and deltamuH indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments.

The equivalent pore radius of intact and damaged mitochondria and the mechanism of active shrinkage

Abstract 1. A procedure is presented which permits the measurement of the equivalent pore radius for the membrane of intact and damaged mitochondria. The approximate values are 6 A for the intact, 11 A for hypotonically treated and KSCN (+ valinomycin)-treated, and 14 A for Ca2+-Pi-treated mitochondria. The values are in accord with the low permeability of the intact and the high permeability of the damaged mitochondria to ions. 2. The inhibitory effect of the polyols on active shrinkage is proportional to the radius of the polyol. 3. The process of active shrinkage is dependent on the concentration and type of electrolyte present in the medium: for cations, Na+ is more active than K+; for anions, the effect follows the Hofmeister series. 4. The osmotic properties of the damaged mitochondria are in accord with the hypothesis that the primary energy-conserving reaction in mitochondrial membranes involves a conformational change and is independent of the development of an osmotic force such as a transmembrane potential.

Permeability to water, dimension of surface, and structural changes during swelling in rat liver mitochondria

SummaryRates and amounts of water translocation across the mitochondrial membrane have been studied with a photometric technique. The process of water translocation can be described in terms of the diffusion equations, and the mitochondria behave as spherical bodies between 15 and 110 mosm. A permeability coefficient to water of 5.3×10−3cm sec−1 has been calculated. The mitochondrial surface is about 1m2/g protein during incubation in 0.10m KCl, and increases to 30 m2/g protein during incubation in 0.005m KCl.The osmotic shrinkage of hypotonically swollen mitochondria has also been studied. Complete reversibility of hypotonic swelling occurs only after incubation of mitochondria in media below 60 to 90 mosm. The appearance of the reversibility is phenomenologically correlated with the rupture of the outer mitochondrial membrane., Below 30 mosm there is a change of the absorbance properties of the membrane. The change correlates with the complete unfolding of the cristae and is attributed to ultrastructural reorganization of the membrane following mechanical stretching.

Distribution of permeant cations in rat liver mitochondria under steady-state conditions.

Abstract The extent of K+ uptake in aerobic mitochondria is dependent on the valinomycin concentration. Also the extent of uptake of organic cations, such as tetrapropylammonium and tetraethylammonium, is dependent on the tetraphenylboron concentration. The results do not support the hypothesis that permeant cations are distributed in mitochondria in the steady state at electrochemical equilibrium and are in accord with a pump and leak mechanism of ion uptake.

Perspectives on the mitochondrial permeability transition

Abstract The permeability transition, a sudden permeability increase of the inner mitochondrial membrane that is greatly favored by Ca2+ accumulation, has puzzled mitochondrial scientists for more than 40 years. It is now recognized that this phenomenon is mediated by opening a high conductance channel (the mitochondrial permeability transition pore) whose open-closed transitions are highly regulated. Through the pore mitochondria may participate in intracellular signalling, and release proteins involved in amplification of the cell death cascade triggered by a variety of physiological and pathological stimuli. Yet, the basic questions of the molecular nature of the permeability transition pore, its physiological role and its very occurrence in vivo remain a matter of intense debate. This short review is meant to summarize our current views on the mitochondrial permeability transition, its perspectives, and our strategies to resolve at least some of the outstanding issues about its nature and function.

H+/site ratio and steady state distribution of divalent cations in mitochondria.

Mitochondria as well as bacteria are known to operate as proton pumps, i.e. the metabolic force is used to transport primarily protons across the membrane. The primary transport of protons is then coupled through various mechanisms to the movement of other species, cations, anions and water. The mechanism of proton transport has been assumed to occur via either respiratory loops [ 1 ] or membrane Bohr effect [2,3] or H’ carrier or channel [4] . Since the membrane Bohr effect and the proton channel may accomodate a stoicheometry of 4 H’/ site while the respiratory loops are compatible with a ratio only of 2 H/site, much interest has been concentrated over the exact determination of the H/site ratio. While Mitchell and Moyle [5 ] reported a ratio of 2 H/site, recently Lehninger and associates [6,7] reported that when the movement of endogenous phosphate is restricted a ratio of 3 or 4 H/site could be measured. This observation supports the earlier conclusions of Azzone and associates [8-lo], who not only measured a ratio of 4 K’/site but also indicated the opportunity of measuring the stoicheometry on cation rather than on H’ charges [ 1 l] . This is due to the masking of the H’ fluxes because of overlapping anion fluxes. A role of endogenous phosphate in affecting the H/site ratio is confirmed also by the appearance of a spin exchange signal in the ESR spectrum during Mr? uptake [ 121; the signal is attributed to (Mn)@O& precipitate. While the view of 4 H/site is gaining increasing consensus, Moyle and Mitchell [ 131 have brought a new argument in favour of 2 H/site, based on the

A quantitative correlation between the kinetics of solutes and water translocation in Liver Mitochondria

SummaryA procedure is described for the calculation of solute fluxes in mitochondria from absorbance measurements. The procedure assumes that mitochondria behave as osmometers and that they are always at osmotic equilibrium.The rates and amounts of K+ translocation have been calculated simultaneously, with the photometric procedure and electrometrically, during passive, K+ efflux coupled to Ca++ uptake and during active K+ uptake and passive K+ release coupled with anion translocation. Good agreement has been found between the two sets of measurements. The data are compatible with the concept that the energy-linked, ion translocation-coupled, mitochondrial swelling is osmotic in nature. It is concluded that the changes of absorbance are quantitatively related to changes in the inner volume and therefore the photometric procedure can be used to calculate, ion fluxes of osmotically active species under various circumstances.

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.

The generation of the proton electrochemical potential and its role in energy transduction

SummaryThe evidence that all energy transducing membranes can generate a proton electrochemical potential difference, Δ