Gf Azzone

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.

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

Proton electrochemical gradient and phosphate potential in submitochondrial particles

The aerobic uptake of inorganic ions, such as 86Rb+ or 125I-, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of deltapH, the transmembrane pH differential, and deltapsi, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the deltapH and deltapsi values from organic and inorganic ions is large at high but not at low ion/protein ratios. 2. In the absence of weak bases and strong acids the values of deltamuH, the proton electrochemical potential difference, are close to 100 mV and the magnitude of deltapH and deltapsi are similar. Weak bases decrease deltapH and enhance deltapsi. Strong acids decrease deltapsi and enhance deltapH. Interchangeability of deltapH with deltapsi occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of deltamuH. 3. Concentrations of weak bases capable of abolishing deltapH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing deltapsi affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of deltapH + deltapsi inhibit ATP synthesis. 4. Depression of deltamuH is paralleled by inhibition of ATP synthesis and decline of deltaGp, the phosphate potential. Abolition of ATP synthesis occurs only when deltamuH is below 20 mV. The deltaGp/deltamuH ratio increases hyperbolically with the decrease of deltamuH.

ESR determination of Mn++ uptake and binding in mitochondria.

Active transport in energy transducing membranes involves the utilization of metabolic energy to move ions against electrochemical gradients. In the case of the divalent cations which are actively transported by mitochondria, namely Ca++, Mn++ and Sr++ [l] , the problem arises as to the determination of the matrix concentration of osmotically active cations. Massari et al. [2] have calculated the ion uptake from absorbancy changes. Gunther and Puskin [3-51 have used the ESR signal of Mn++ to distinguish between free and bound cations and to calculate the matrix concentration of free Mn++. According to Gunther and Puskin [3], during active uptake the Mn++ spectrum can be split into two components E and S belonging to membrane bound and matrix free Mn++, respectively. The binding of divalent cations to the membrane has also been followed qualitatively through the enhancement of the fluorescent chelate of tetracycline with divalent cations [6]. In the present paper the changes of e.s.r. signal of Mn++ during active uptake have been correlated with absorbance and.rhatrix volume changes. On the basis of the combined volume and e.s.r. data a procedure is described for the determination of free Mn++ concentration in the mitochondrial matrix.

Nature of respiratory stimulation in hyperthyroidism: the redox behaviour of cytochrome c

Hyperthyroid mitochondria show an increased K m and V max in the high affinity phase of cytochrome oxidase kinetics. During inhibitor titrations, cytochrome c shows a different redox behaviour in hyperthyroid with respect to protonophore‐treated euthyroid mitochondria. The observations are discussed in terms of a different regulation of electron input and output into the respiratory chain during slip and leak types of uncoupling. In hyperthyroid mitochondria during inhibitor titrations, the pattern of the relationship between uncoupler‐induced extra‐respiration and membrane potential is highly non‐linear. The complex nature of the respiratory stimulation in hyperthyroid mitochondria is discussed.

On the nature of the uncoupling effect of fatty acids

The effect of palmitic acid on the electrical potential difference Δψ across the inner mitochondrial membrane appears to depend on the medium in which mitochondria are incubated. In medium A (cf. Luvisettoet al. (1987),Biochemistry,26, 7332–7338) Δψ decreases much more than in medium B (cf. Rottenberg and Hashimoto (1986),Biochemistry,25, 1747–1755) at concentrations of fatty acid which equally stimulate the rate of respiration in state 4. Valinomycin and NaCl were both present in medium B and absent in medium A. However, in both media the pattern of the P/O ratio as a function of antimycin in the presence of a constant amount of palmitic acid or of FCCP shows similar behaviour. We conclude that in both media palmitic acid increases the membrane conductance to protons, but for unclear reasons the Δψ assay fails to measure the decline of Δψ in medium B. However, the increase in membrane conductance induced by palmitic acid does not quantitatively account for the stimulation of the rate of respiration.

The nature of uncoupling by n-hexane, 1-hexanethiol and 1-hexanol in rat liver mitochondria

We have analyzed the effects of n-hexane, 1-hexanethiol, and 1-hexanol on the coupled respiration of rat liver mitochondria. Incubation of mitochondria with n-hexane, 1-hexanethiol and 1-hexanol resulted in a stimulation, at low concentrations, and an inhibition, at high concentrations, of the state 4 mitochondrial respiration. Three criteria, all based on the comparison with the effect of DNP, have been used to establish whether the stimulation of respiration, at low concentrations of n-hexane, 1-hexanethiol, and 1-hexanol, depends on protonophoric mechanisms. First, the quantitative relationship between the extents of respiratory stimulation and membrane potential depression: a strong decrease of membrane potential was induced by increasing concentrations of DNP and a negligible depression by increasing concentrations of n-hexane or 1-hexanethiol. Only a slight decrease was induced by 1-hexanol. Second, the quantitative relationship between the extents of respiratory stimulation and of proton conductance increase: at equivalent rates of respiration, the enhancement of the proton conductance induced by DNP was very marked, by n-hexane and 1-hexanethiol practically negligible, and by 1-hexanol much smaller than that induced by DNP. Third, in titrations with redox inhibitors of the proton pumps, the pattern of the relationship between proton pump conductance and membrane potential was markedly different from protonophoric and non-protonophoric uncouplers: almost linear in the case of DNP, highly non-linear in the case of n-hexane, 1-hexanethiol and 1-hexanol. These three criteria support the view that n-hexane, 1-hexanethiol, and partially 1-hexanol, uncouple mitochondrial respiration by a non-protonophoric mechanism.

Functional and structural changes induced by phospholipase C in intact mitochondria and submitochondrial particles

Abstract 1. (1) Phospholipase C from Bacillus cereus causes uncoupling both in intact mitochondria and sonic particles. The uncoupling effect in intact mitochondria occurs parallel to the loss of osmotic properties, while in particles there is an inhibition of membrane bound enzymes. The kinetics of phospholipid hydrolysis and uncoupling are preceeded by a lag phase in mitochondria but not in particles. 2. (2) The extent of phospholipid hydrolysis by acid-base titratin is 55% and 43% in mitochondria and particles, respectively. Chemical analysis gives an extent of hydrolysis of 38% and 30%, respectively. The degree of hydrolysis of phosphatidylcholine and phosphatidylethanolamine is the same in mitochondria and particles. Cardiolipin is not hydrolyzed. 3. (3) Fatty acids with spin label near the phospholipid polar heads indicate the formation of fluid hydrophobic regions in mitochondria. Also the pyrene fluorescence indicates an increased fluidity in mitochondria. In addition the phospholipase C attack is accompanied both in mitochondria and particles by an apparent increased immobilization of the spin label. 4. (4) The data are discussed in terms of the structural differences between mitochondria and particles and of a dual role of phospholipids in controlling enzymatic activities and osmotic properties.

Effect of P-Lipase C on the Structure and Function of Mitochondria and Sonic Fragments

The purpose of this study is to illustrate some data on the use of P-lipase C to elucidate the membrane structure of intact mitochondria and submitochondrial particles (1,2). Fig. 1 shows the effect of P-lipase C from B. cereus on the fluorescence of mitochondrial NADH (3). The fluorescence is maximal when the mitochondria are in a tightly coupled state and decreases parallel to uncoupling. The extent of uncoupling increases proportionally to the amount of P-lipase C and occurs always after a lag phase. In the same Figure it is also shown the rapid abolition of the fluorescence caused by the powerful uncoupler FCCP.