Giovanni Felice Azzone

Determination of the Proton Electrochemical Gradient across Biological Membranes

Publisher Summary This chapter explains the ion distribution method for the determination of Δ ψ and ΔpH, while other approaches are analyzed only in comparison with this method. The limitation in choice for discussion is that ion distribution is the only method suited for a theoretical treatment. Other approaches analyze the absorbance and fluorescence changes accompanying variations of Δ ψ and ΔpH in the presence of various probes, either extrinsic or intrinsic. The spectroscopic techniques are used quantitatively only after calibrations with the ion distribution method. These considerations do not apply to the assessment of ΔpH by P NMR or to the determination of Δ ψ and ΔpH by microelectrodes. However, the former technique is highly specialized and requires extremely high protein concentrations, and the latter is of limited applicability to organelles of small dimensions and is not routinely available. The chapter also discusses problems that may benefit from accurate Δψ H measurements, including the determination of the stoichiometries of the H + pumps and the assessment of Δψ H as the coupling intermediate between the two H + pumps

Phenylarsine oxide induces the cyclosporin a-sensitive membrane permeability transition in rat liver mitochondria

This paper reports an investigation on the effects of the hydrophobic, bifunctional SH group reagent phenylarsine oxide (PhAsO) on mitochondrial membrane permeability. We show that PhAsO is a potent inducer of the mitochondrial permeability transition in a process which is sensitive to both the oxygen radical scavanger BHT and to cyclosporin A. The PhAsO-induced permeability transition is stimulated by Ca2+ but takes place also in the presence of EGTA in a process that maintains its sensitivity to BHT and cyclosporin A. Our findings suggest that, at variance from other known inducers of the permeability transition, PhAsO reacts directly with functional SH groups that are inaccessible to hydrophilic reagents in the absence of Ca2+.

Safranine as membrane potential probe in rat liver mitochondria.

Abstract The absorbance changes accompanying safranine uptake due either to respiration or to K + diffusion potential are maximal at a point of equivalence between number of dye molecules and binding sites (“saturation” point) and decrease both below and above this point. This behavior is in accord with a model where safranine uptake is followed by stacking on adjacent membrane sites below saturation and destacking on nonadjacent sites above saturation. The values of the endpoint titrations are similar for the respiration- and K + diffusion potentialinduced safranine uptakes only at protein concentrations above saturation. Calibration of the safranine response with K + diffusion potentials indicates a linear region at low Δψ and a deviation from linearity at high Δψ. The region of linear safranine response depends on the dye/protein ratio. Increase of dye/protein ratio leads to expansion of the linear range. However calibration at high dye/protein ratios is limited by the loss of matrix K + . At constant dye/protein ratios increase of dye concentration shifts the calibration curve at lower Δψ values. The half-time of the safranine response increases hyperbolically with the decrease of protein at constant dye concentration and with the decrease of dye concentration at constant dye/protein ratios. Uptake and binding of safranine result in mitochondrial damage with respect to degree of energy coupling, rate of electron transport, and ADP-stimulated respiration. Oxygen pulses to anaerobic mitochondria result in an electrical field which may be followed with the safranine response. The minimal amount of oxygen capable of inducing a maximal rate of safranine uptake is 0.5 natom × mg protein −1 . Since the rate of safranine uptake is linear with Δψ, this amount of oxygen corresponds to the extent of charge separation required for a steady state Δψ.

Kinetics of calcium(2+) ion carrier in rat liver mitochondria

The rate of aerobic Ca2+ transport is limited by the rate of the H+ pump rather than by the Ca2+ carrier. The kinetics of the Ca2+ carrier has therefore been studied by using the K+ diffusion potential as the driving force. The apparent Vmax of the Ca2+ carrier is, at 20 degrees C, about 900 nmol (mg of protein)-1 min-1, more than twice the rate of the H+ pump. The apparent Vmax is depressed by Mg2+ and Li+. This supports the view that the electrolytes act as noncompetitive inhibitors of the Ca2+ carrier. The degree of sigmoidicity of the kinetics of Ca2+ transport increases with the lowering of the temperature and proportionally with the concentration of impermeant electrolytes such as Mg2+ and Li+ but not choline. The effects of temperature and of electrolyte do not support the view that the sigmoidicity is due to modifications of the surface potential. Rather, they suggest that Ca2+ transport occurs through a multisubunit carrier, where cooperative phenomena are the result of ligand-induced conformational changes due to the interaction of several allosteric effectors with the carrier subunits. In contrast with La3+ which acts as a competitive inhibitor, Ruthenium Red affects the kinetics by inducing phenomena both of positive and of negative cooperativity. The Ruthenium Red induced kinetics has been reproduced through curve-fitting procedures by applying the Koshland sequential interaction hypothesis to a four-subunit Ca2+ carrier model.

Permeability of inner mitochondrial membrane and oxidative stress

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.

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.

Swelling and shrinkage phenomena in liver mitochondria VI. Metabolism-independent swelling coupled to ion movement

Abstract 1. Water uptake coupled to ion movement has been studied in respiratory-inhibited liver mitochondria, of which the permeability to cations was increased by valinomycin, gramicidin or EDTA, and to anions by raising the pH of the medium. The movement of water was accounted for by the osmotic pressure of the penetrating solutes. 2. The rate of movement of water was inversely proportional to the concentration of solutes in the medium, and was dependent on the presence of permeating cations and anions. The above findings are interpreted within the concept of an osmotic movement of water. 3. The flow of anions through the membrane was inhibited by Ca 2+ and Mn 2+ Mitochondrial swelling was inhibited by sucrose. 4. Ion movement was independent of energy supply from metabolism. The nature of the force driving the ion movement is discussed.

Mosaic protonic coupling hypothesis for free energy transduction

*B.C.P. Jansen Instituut, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands and Istituto di Chimica Biologica, Universita di Padova, Via F. Marzolo 3, 35100 Padova, +Istituto ed Orto Botanico, Universita di Bologna, Via Irnerio 42, 40126 Bologna, ?Dipartimento di Fisica, Universita della Calabria, 87036 Cosenza, **C.N.R. Unit for Physiology of Mitochondria, Universita di Padova, Via Loredan 16, 35100 Padova, Italy and ++Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, Wales

Proton electrochemical potential in steady state rat liver mitochondria.

Delta approximately muH has been determined in steady state mitochondria by measuring the magnitude of delta pH on the distribution of acetate and of deltapsi on the distribution of K+, tetraphenylphosphonium, Ca2+, Sr2+ and Mn2+. (1) The matrix concentration of divalent cations has been calculated from the total cation uptake, from the increase of matrix volume and from the ESR sextet signal of Mn(H2O)L2+. The [cat2+]i based on osmotic data is about five times higher than that based on ESR measurements. The [cat2+]i based on total uptake is much higher than that based on osmotic data at low cation/protein ratios. (2) In the presence of 10 mM acetate the maximal deltapsi on Ca2+ is about 130 mV and on Sr2+ is 95 mV. Deltapsi on Mn2+ is 91 or 109 mV, according to whether [cat2+-a)i is calculated from ESR or osmotic data. Under the same conditions, deltapH is about 60 mV. Hence delta approximately muH on divalent cations is between 151 and 190 mV. (3) Deltapsi on K+, in valinomycin treated mitochondria with 10 mM acetate or 2 mM Pi, drops from 200 mV, at low [K+]0 to almost zero parallel to the increase of [K+]0. DeltapH is 30 mV at low [K+]0 and about 42 mV at 600 muM K+. Hence delta approximately muH drops from 22m mV lower values with the increase of [K+]0. (4) Maximal deltapsi on triphenylmethylphosphonium is 140 mV. (5) When delta approximately muH is measured simultaneously on divalent cations and on K+, the values on K+ tend to approach those on Ca2+ while those on Sr2+ are about 50 mV lower. (6) It is concluded that the steady state mitochondrial energy potential is equivalent to a delta approximately muH between 150 and approx. 190 mV.

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.