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G F Azzone

Publications and source records attributed to G F Azzone.

At least 55 records · Page 3Linked to original sources
PubMed1986

Intrinsic uncoupling of mitochondrial proton pumps. 1. Non-ohmic conductance cannot account for the nonlinear dependence of static head respiration on delta microH.

The passive membrane conductance LH1 of rat liver mitochondria has been measured and compared with the quantity nJesh/delta microHsh (n = H+/e stoichiometry; Jesh = rate of electron transfer in static head) over a delta microH range. The two curves approach each other only in the lower part of the range, while they sharply diverge at large values of delta microH. Thus nJesh/delta microHsh cannot be considered to be a measure of LH1 in the upper delta microH region. Only a fraction of the static head electron flow is accounted for by futile proton cycling via leaks. Contaminating open membrane fragments or completely leaky mitochondria can be responsible for only a small part of the residual rate of oxygen consumption. We conclude that a large part of static head respiration must have yet another cause and propose it to be intrinsic uncoupling of the respiratory chain enzymes.

Animals↗
PubMed1986

Intrinsic uncoupling of mitochondrial proton pumps. 2. Modeling studies.

The thermodynamic and kinetic properties associated with intrinsic uncoupling in a six-state model of a redox proton pump have been studied by computing the flow-force relations for different degrees of coupling. Analysis of these relations shows the regulatory influence of the thermodynamic forces on the extent and relative contributions of redox slip and proton slip. Inhibition has been introduced into the model in two different ways, corresponding to possible modes of action of experimental inhibitors. Experiments relating the rate of electron transfer to delta microH at static head upon progressive inhibition of the pumps have been simulated considering (1) the limiting case that the nonzero rate of electron transfer at static head is only due to intrinsic uncoupling (no leaks) and (2) the experimentally observed case that about 30% of the nonzero rate of electron transfer at static head is due to a constant proton leakage conductance in parallel with the pumps, the rest being due to intrinsic uncoupling. The same simulations have been performed for experiments in which the rate of electron transfer is varied by varying the substrate concentration rather than by using an inhibitor. The corresponding experimental results obtained by measuring delta microH and the rate of electron transfer at different succinate concentrations in rat liver mitochondria are presented. Comparison between simulated behavior and experimental results leads to the general conclusion that the typical relationship between rate of electron transfer and delta microH found in mitochondria at static head could certainly be a manifestation of some degree of intrinsic uncoupling in the redox proton pumps.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

A minimal hypothesis for membrane-linked free-energy transduction. The role of independent, small coupling units.

Experimental data are reviewed that are not in keeping with the scheme of 'delocalized' protonic coupling in membrane-linked free-energy transduction. It turns out that there are three main types of anomalies: (i) rates of electron transfer and of ATP synthesis do not solely depend on their own driving force and on delta mu H, (ii) the ('static head') ratio of delta Gp to delta mu H varies with delta mu H and (iii) inhibition of either some of the electron-transfer chains or some of the H+-ATPases, does not cause an overcapacity in the other, non-inhibited proton pumps. None of the earlier free-energy coupling schemes, alternative to delocalized protonic coupling, can account for these three anomalies. We propose to add a fifth postulate, namely that of the coupling unit, to the four existing postulates of 'delocalized protonic coupling' and show that, with this postulate, protonic coupling can again account for most experimental observations. We also discuss: (i) how experimental data that might seem to be at odds with the 'coupling unit' hypothesis can be accounted for and (ii) the problem of the spatial arrangement of the electrical field in the different free-energy coupling schemes.

ATP Synthetase Complexes↗
PubMed1984

Pathway for uncoupler-induced calcium efflux in rat liver mitochondria: inhibition by ruthenium red.

The rate of uncoupler-induced Ca2+ efflux from rat liver mitochondria is increased by acetate and decreased by phosphate. This effect depends on a shift of the apparent Km, which is increased by phosphate and decreased by acetate, while the Vmax is not modified. The modification of the apparent Km by permeant anions presumably reflects changes in the concentration of matrix free Ca2+. A major part of uncoupler-induced Ca2+ efflux is sensitive to Ruthenium Red, the specific inhibitor of the Ca2+ uniporter , but an apparent insensitivity is observed when the H+ permeability is rate limiting in the process of Ca2+ efflux. The titer of uncoupler required for maximal stimulation of Ca2+ efflux increases with the Ca2+ load and may be 1-2 orders of magnitude higher than that required for maximal stimulation of respiration. On the other hand, when the uncoupler concentration is raised above 10(-6) M, the process of Ca2+ efflux becomes again Ruthenium Red insensitive. The Ruthenium Red inhibition of uncoupler-induced Ca2+ efflux is time dependent, in that the degree of inhibition exerted by low amounts of Ruthenium Red increases with the incubation time. Since the inhibition of the rate of Ca2+ influx undergoes a parallel relief, it is possible that Ruthenium Red moves from the cytosolic to the matrix side of the inner membrane. It is concluded that, in native mitochondria, uncoupler-induced Ca2+ efflux occurs via reversal of the uniport Ca2+ carrier, and not through activation of an independent pathway.

Animals↗
PubMed1983

Electroneutral H+-K+ exchange in liver mitochondria. Regulation by membrane potential.

The paper analyzes the factors affecting the H+-K+ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg2+ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K+ efflux following addition of A23187 in low-K+ media. (1) The H+-K+ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of delta pH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, delta psi. Maximal rate of K+ efflux is observed at 180-190 mV, whereas K+ efflux is inhibited below 140-150 mV. (2) Activation of H+-K+ exchange leads to depression of delta pH but not of delta psi. Respiration is only slightly stimulated by the onset of H+-K+ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the delta psi control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H+-K+ exchange, while leaving unaffected the level of Mg2+ depletion. This type of activation results in partial 'uncoupling' from the delta psi control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high delta psi. (4) The available evidence suggests that the activity of the H+-K+ exchanger is modulated by the electrical field across the inner mitochondrial membrane.

Adenosine Triphosphatases↗
PubMed1983

Regulation of Ca2+ efflux in rat liver mitochondria. Role of membrane potential.

The paper analyzes the relationship between membrane potential (delta psi), steady state pCao (-log [Ca2+] in the outer aqueous phase) and rate of ruthenium-red-induced Ca2+ efflux in liver mitochondria. Energized liver mitochondria maintain a pCao of about 6.0 in the presence of 1.5 mM Mg2+ and 0.5 mM Pi. A slight depression of delta psi results in net Ca2+ uptake leading to an increased steady state pCao. On the other hand, a more marked depression of delta psi results in net Ca2+ efflux, leading to a decreased steady-state pCao. These results reflect a biphasic relationship between delta psi and pCao, in that pCao increases with the increase of delta psi up to a value of about 130 mV, whereas a further increase of delta psi above 130 mV results in a decrease of pCao. The phenomenon of Ca2+ uptake following a depression of delta psi is independent of the tool used to affect delta psi whether by inward K+ current via valinomycin, or by inward H+ current through protonophores or through F1-ATP synthase, or by restriction of e- flow. The pathway for Ca2+ efflux is considerably activated by stretching of the inner membrane in hypotonic media. This activation is accompanied by a decreased pCao at steady state and by an increased rate of ruthenium-red-induced Ca2+ efflux. By restricting the rate of e- flow in hypotonically treated mitochondria, a marked dependence of the rate of ruthenium-red-induced Ca2+ efflux on the value of delta psi is observed, in that the rate of Ca2+ efflux increases with the value of delta psi. The pCao is linearly related to the rate of Ca2+ efflux. Activation of oxidative phosphorylation via addition of hexokinase + glucose to ATP-supplemented mitochondria, is followed by a phase of Ca2+ uptake, which is reversed by atractyloside. These findings support the view that Ca2+ efflux in steady state mitochondria occurs through an independent, delta psi-controlled pathway and that changes of delta psi during oxidative phosphorylation can effectively modulate mitochondrial Ca2+ distribution by inhibiting or activating the delta psi-controlled Ca2+ efflux pathway.

Animals↗
PubMed1983

The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver.

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.

Adenosine Triphosphate↗
PubMed1983

Molecular slipping in redox and ATPase H+ pumps.

The titration of the mitochondrial ATPase H+ pump with oligomycin has been compared with the titration of the redox H+ pump with antimycin. In both cases there is extensive inhibition of the pumps without significant depression of delta muH. The two pumps exhibit 'nonohmic' behavior in different ranges of delta muH. This discrepancy favors the hypothesis of nontightly coupled or 'slipping' H+ pumps with respect to that of a steep dependence of the membrane 'leak' conductance for H+ on delta muH.

Adenosine Triphosphatases↗
PubMed1983

Studies on the relationship between ATP synthesis and transport and the proton electrochemical gradient in rat liver mitochondria.

The effect of ATP synthesis on delta mu H in rat liver mitochondria has been analyzed by separating the steps of adenine nucleotide translocation and ATP synthesis in the matrix. Either exchange of ATP, synthesized by substrate level phosphorylation in the matrix of oligomycin-treated mitochondria, for external ADP, or activity of the membrane-bound ATP synthase complex results in delta mu H depression with respect to resting state levels. This depression appears to be more pronounced, under strictly comparable conditions, when arsenate is used to stimulate ATP synthase activity than when the ornithine-citrulline conversion reaction is used for the same purpose.

Adenosine Triphosphatases↗
PubMed1982

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria.

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.

Adenosine Triphosphate↗
PubMed1982

Activation of site I redox-driven H+ pump by exogenous quinones in intact mitochondria.

The site I redox-driven H+ pump has been activated by the addition of exogenous quinones to antimycin A-KCN-inhibited mitochondria. The rate of quinone reduction and the degree of rotenone sensitivity increase in the order, duroquinone less than ubiquinone0 less than ubiquinone1. Apparent Km, Vmax, and degree of sigmoidicity during e- transfer in the absence and presence of rotenone have been determined for each quinone. The data support the view that the NADH dehydrogenase possesses two redox sites, one accounting for the rotenone-sensitive reduction and another accounting for the rotenone-insensitive reduction. The degree of activation of the redox H+ pump, which reflects the rotenone-sensitive e- transfer, depends, for each quinone, on the relative Km, Vmax, and sigmoidicity of the rotenone-sensitive and insensitive processes. The redox H+ pump activation is highest with ubiquinone1, where the rotenone-sensitive reaction has a lower Km than that of the rotenone-insensitive reaction, and lowest with duroquinone where the rotenone-insensitive reaction has a high Vmax and no sigmoidicity with respect to that of the rotenone-sensitive reaction. Using ubiquinone1 the stoichiometry of the site I redox-driven H+ pump has been determined on either the flow or the force ratios. The flow ratios approached values of 4 H+/2 e- under conditions close to stationary state for H+ pumping and to zero for H+ electrochemical gradient. The force ratio also approached values close to 4 H+/2 e- under static head conditions.

Adenosine Triphosphatases↗
PubMed1982

ATP synthesis during exogenous NADH oxidation. A reappraisal.

This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c.

Adenosine Triphosphatases↗
PubMed1982

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.

Animals↗
PubMed1982

Non-equilibrium thermodynamic assessment of redox-driven H+ pumps in mitochondria.

Isolated mitochondria suspended in an aerobic medium with 3-hydroxybutyrate or succinate serving as electron donor attain a stationary state with vanishing net flow of H+ ions (state 4). Adding valinomycin to such a suspension in the presence of various concentrations of K+ ions and a weak acid system such as acetate or phosphate creates new stationary states for the mitochondria which are characterized by a constant influx of K+ ions, while the net flow of H+ ions again vanishes due to the recycling of these ions by the weak acid system. Sufficiently low concentrations of K+ ions (less than 4 mM) cause these stationary states to last long enough for a separation of the mitochondria by centrifugation. The difference in electrochemical potential for H+ ions can then be determined by means of the partitioning of radioactively labelled markers. Suitable procedures to correct for binding of the markers are described. It is found that, for a constant affinity of the electron in the suspending medium, electron flow and the flow of K+ ions, which indicates the flow of pumped H+ ions, are linearly dependent on the electrochemical potential difference of H+ ions. The phenomenological coefficients obtained from these correlations are discussed with respect to the contributions of additive constants in the linear relations. It is found that, under the present experimental condition, such constants most likely vanish thus yielding symmetric flow-force relations. It is concluded that the redox-driven H+ pumps are not tightly coupled due to molecular slipping in the pumps and that the molecular stoichiometry is 2 H+ ions/electron for coupling site I and 4 H+ ions/electron for coupling sites II and III together.

Animals↗