1,721,003 research outputs found
Regulation of Ca2+ Efflux in Rat Liver Mitochondria: Role of Membrane Potential
No full textThe 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
Cytochrome c as an Electron Shuttle Between the Outer and Inner Mitochondrial Membranes
No full textAddition of exogenous NADH to rotenone- and antimycin A-treated mitochondria, in 125 mM KCl, results in rates of oxygen uptake of 0.5-1 and 10-12 nanoatoms of oxygen X mg protein-1 X min-1 in the absence and presence of cytochrome c, respectively. During oxidation of exogenous NADH there is a fast and complete reduction of cytochrome b5 while endogenous or added exogenous cytochrome c become 10-15% and 100% reduced, respectively. The reoxidation of cytochrome b5, after exhaustion of NADH, precedes that of cytochrome c. NADH oxidation is blocked by mersalyl, an inhibitor of NADH-cytochrome b5 reductase. These observations support the view of an electron transfer from the outer to the inner membrane of intact mitochondria. Both the rate of exogenous NADH oxidation and the steady state level of cytochrome c reduction increase with the increase of ionic strength, while the rate of succinate oxidation undergoes a parallel depression. These observations suggest that the functions of cytochrome c as an electron carrier in the inner membrane and as an electron shuttle in the intermembrane space are alternative. It is concluded that aerobic oxidation of exogenous NADH involves the following pathway: NADH leads to NADH-cytochrome b5 reductase leads to cytochrome b5 leads to intermembrane cytochrome c leads to cytochrome oxidase leads to oxygen. It is suggested that the communication between the outer and inner membranes mediated by cytochrome c may affect the oxidation-reduction level of cytosolic NADH and the related oxidation-reduction reactions
Electroneutral H+/K+ Exchange in Liver Mitochondria: Regulation by Membrane Potential
No full textThe 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
ATP Synthesis During Exogenous NADH Oxidation: a Reappraisal
No full textThis 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
DeltapH-induced Ca2+ Fluxes in Rat Liver Mitochondria
No full text1. The paper reports an investigation on ΔpH-driven Ca2+ fluxes in rotenone-treated rat liver mitochondria. The H+ concentration gradient (acidic inside) required to drive Ca2+ influx is obtained from the K+ concentration gradient through a H+/K+ exchange. The rate of H+/K+ exchange is very low in native mitochondria and is markedly enhanced by the electroneutral ionophore nigericin. The rate of Ca2+ influx depends on the rate of generation of ΔpH; it is negligible in native mitochondria and fast after nigericin.
2. The process of ΔpH-driven Ca2+ influx shows three basic features. First, the rate of Ca2+ influx is markedly increased by the addition of uncouplers. Second, the rate of Ca2+ influx is inhibited by ruthenium red with the same sensitivity as the processes driven by the H+ pump or K+ diffusion. The electroneutral H+/Ca2+ ionophore A23187 restores Ca2+ influx in mitochondria inhibited by ruthenium red. Third, both rate and extent of ΔpH-driven Ca2+ influx are inhibited by the addition of weak bases but not of weak acids.
3. The process of ΔpH-driven Ca2+ influx is accompanied by H+ efflux. A correlation also exists between generation of ΔpH (acidic inside) due to H+/K+ exchange and utilization of ΔpH due to H+/Ca2+ exchange. The process of ΔpH-driven, A23187-catalyzed, H+/Ca2+ exchange is not preceeded by rapid H+/K+ exchange in the absence of nigericin.
4. After termination of the phase of Ca2+ influx a phase of Ca2+ efflux ensues. Ca2+ efflux is not coupled to H+ or K+ reentry and is completely inhibited by ruthenium red. Ca2+ efflux is not observed when Ca2+ transport is catalyzed by A23187 in mitochondria inhibited by ruthenium red. Furthermore A23187 abolishes Ca2+ efflux also when Ca2+ influx occurs in the absence of ruthenium red.
5. The data suggest that the ΔpH-driven Ca2+ influx and the subsequent Ca2+ efflux take place through the native carrier as electrical uniport
Activation of site I redox-driven H+ pump by exogenous quinones in intact mitochondria.
No full textThe 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
Synthesis of ATP During Oxidation of Exogenous NADH by Intact Liver Mitochondria: a Reappraisal
No full textEffect of funiculosin and antimycin A on the redox-driven H+-pumps in mitochondria: on the nature of "leaks'.
No full textThe effect of antimycin A and funiculosin, two inhibitors which block electron transfer in the b-c1 complex, on electron flow and electrochemical potential difference of H+ ions in mitochondria at static head (state 4) is investigated. In addition, the respiratory control ratio is determined as the ratio between uncoupler stimulated and static-head electron flow. Malonate, a competitive inhibitor or succinic dehydrogenase, is used for comparison. All three inhibitors cause an extensive depression of static-head electron flow but only a limited decrease in the electrochemical potential difference of H+ ions. With the antimycin-type of inhibitors, the respiratory control ratio slightly increases up to about 50% inhibition of electron flow and then steeply declines. With malonate, a strong decrease of the respiratory control ratio is observed in a concentration range where the electron flow is inhibited less than 10%. It is shown than the data do not comply with the generally accepted hypothesis of a leak conductance being regulated by the electrochemical potential difference of H+ ions. They can be interpreted in terms of not tightly coupled redox-driven H+-pumps. A non-vanishing electron flow at static head then arises predominantly from molecular slipping in the pumps, and the (constant) leak conductance yields only a minor contribution
Analysis of mechanisms of free-energy coupling and uncoupling by inhibitor titrations: theory, computer modeling and experiments.
No full textThe rates of ATP synthesis and of ATP-driven NAD reduction have been measured in bovine heart submitochondrial particles as a function of the fraction of inhibited redox pumps (in titrations with either antimycin or rotenone) and of the fraction of inhibited ATPases (in titrations with DCCD). The flux control coefficients of the redox and ATPase proton pumps on the rates of ATP synthesis and of ATP-driven NAD reduction have been derived and found to be equal to 1 for both pumps; i.e., both pumps appear to be 'completely rate limiting'. A theoretical analysis of the inhibitor titration approach based on kinetic models of chemiosmotic coupling and on the theory of metabolic control is presented. This analysis (i) shows that the results of the single inhibitor titrations are incompatible with a delocalized chemiosmotic mechanism of energy coupling if the proton conductance of the membrane is sufficiently low with respect to the conductances of the pumps; and (ii) suggests an experimental approach based on the determination of the P/O and the respiratory control ratios at different degrees of inhibition of the proton pumps to establish the origin of the 'loose coupling' of submitochondrial particle preparations. Three independent types of observation show that the 'loose coupling' of the particle preparation is not mainly due to an increased membrane proton conductance. The same and other independent observations are consistent with the view that the loose coupling of submitochondrial particle preparation is due mainly to inhomogeneity, i.e. to the presence of a subpopulation of highly leaky non-phosphorylating vesicles respiring at maximal rate. The results as a whole together with the simulations and analysis presented lead to the conclusion that the mechanism of free-energy coupling in submitochondrial particles is not completely delocalized
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