Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.     

The effect of tetraphenylborate on mitochondrial energy transduction.

In phosphorylating submitochondrial particles, tetraphenylborate binds to the specific uncoupler binding site and inhibits oxidative phosphorylation, ATP-P_i exchange and ATP-driven reverse electron transport. In contrast, intact mitochondria are unaffected in uncoupler binding and energy transfer at the concentrations used in submitochondrial particles. The proton permeability of submitochondrial particles is only slightly increased (10–20%) at concentrations of tetraphenylborate which cause 50% uncoupling (4–8 μM). These results, and those obtained earlier with picrate, are consistent with a three-step mechanism of uncoupling which involves binding of uncoupler anions, protonation and dissociation of the resulting neutral uncoupler molecule.     

Relation of superoxide generation and lipid peroxidation to the inhibition of NADH-Q oxidoreductase by rotenone, piericidin A, and MPP+.

The addition of NADH to submitochondrial particles inhibited by agents which interrupt electron transport from NADH-Q oxidoreductase (Complex I) to Q10 (rotenone, piericidin A, and MPP+) results in superoxide formation and lipid peroxidation. A study of the quantitative relations now shows that oxyradical formation does not appear to be the direct result of the inhibition. Although tetraphenyl boron (TPB) greatly enhances the inhibition by MPP+, it has no effect on O2. formation or lipid peroxidation. When submitochondrial particles completely inhibited by rotenone or piericidin A are treated with bovine serum albumin to remove spuriously bound inhibitor molecules without affecting those bound at the specific inhibition site, NADH-Q activity remains inhibited and lipid peroxidation occurs but superoxide formation ceases. Thus oxyradical formation may be the result of the binding of inhibitors at sites in the membrane other than those related to the inhibition of electron transport.     

Anisotropic action of cetyl pyridinium chloride on rat heart mitochondria

This work reports experiments that show that in rat heart mitochondria, the alkyl cation cetyl pyridinium chloride induces inhibition of the electron transport with NAD-dependent substrates. It also induces an enhancement of oxygen uptake with succinate as substrate, stimulation of adenosine triphosphatase activity, release of Ca²⁺ that have been accumulated, and inhibition of the energy-dependent uptake of ethidium bromide; these findings suggest that cetyl pyridinium chloride induces a collapse of membrane potential. The experiments carried out with submitochondrial particles showed that this reagent inhibits the oxidation of NADH, provided an uncoupler is added to the system. According to these data it is proposed that the latter effect is due to the binding of cetyl pyridinium chloride to the inner mitochondrial membrane in a site that faces the cytosol.     

The interaction of the potential-sensitive molecular probe merocyanine 540 with phosphorylating beef heart submitochondrial particles under equilibrium and time-resolved conditions

The interaction of the potential-sensitive extrinsic molecular probe merocyanine 540 ( M540 ) with phosphorylating submitochondrial particles has been investigated under equilibrium and time-resolved conditions. The addition of ATP to a M540 -membrane suspension produces oligomycin and CCCP-sensitive spectral changes with absolute maxima near 490, 530, and 565 nm; a 1- to 2-nm red shift of the dye absorption spectrum is also evident in the longer-wavelength region of the spectrum. In fixed-wavelength work, the energy-dependent optical signals were increased by the addition of nigericin and NH4Cl, and could be subsequently restored to the control level by valinomycin or KSCN, respectively. These observations suggest that M540 is specifically sensitive to the membrane-potential portion of the electrochemical gradient presumably present in the submitochondrial particle system in the presence of substrate. Binding analyses based on the Langmuir adsorption isotherm and the direct linear method indicate that the M540 dissociation constant is decreased by the presence of ATP with little or no change in the maximum number of binding sites. The M540 dissociation constant was markedly decreased when 0.1 M NaCl was present in the medium, suggesting that the association of this probe with the membrane may be subject to considerable surface charge repulsion. Results from the binding analyses indicate that the origin of the energy-dependent spectral changes may be an enhanced association of M540 with the submitochondrial particle membrane resulting from the transfer of dye from the aqueous phase to membrane-binding sites. The time course of the NADH-, ATP-, or succinate-induced signal developed slowly, on a time scale of tens of seconds, and follows a second-order rate law, suggesting that the rate-limiting step in the development of the ATP-induced M540 signal may be the transfer of dye from the aqueous phase to membrane-binding sites. The enhanced passive binding of M540 to the submitochondrial particle membrane in the presence of NaCl reduces the concentration of free dye apparently available to redistribute to the membrane when substrate is present, with a concomitant reduction in the observed pseudo-first-order and the second-order rate constants. If the effective free dye concentration is estimated from binding data and used in the plot from which the latter rate constant is obtained, the value of this constant compares favorably with the obtained in the absence of the electrolyte.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of totally inverted submitochondrial particles by sonication of beef heart mitochondria

A novel procedure for isolating totally inverted preparations of submitochondrial particles by sonication of beef heart mitochondria is described. The procedure involves only differential centrifugation in 0.25 M sucrose containing 0.15 M KCl. The submitochondrial particles have 96% of their cytoplasmic face cytochromec-binding sites sequestered within the particles. Mild sonication exposes cytochromec-binding sites to the medium. The oligomycin-sensitive ATPase of sonic-derived submitochondrial particles, like that of electron transport particles, is inhibited 98% by exogenous isolated ATPase inhibitor protein. NADH oxidase activity in these particles is inhibited by oligomycin. The respiratory control index (uncoupled rate/oligomycin-inhibited rate) is approximately 3.4 and can be increased by washing the particles with medium containing bovine serum albumin.     

Platanetin and 7-iodo-acridone-4-carboxylic acid are not specific inhibitors of respiratory NAD(P)H dehydrogenases in potato tuber mitochondria

Progress in understanding the role of NAD(P)H oxidation in plant respiration is restricted by the lack of access to specific inhibitors of each of the unknown number of NAD(P)H dehydrogenases in the inner mitochondrial membrane. Platanetin (3,5,7,8-tetrahydroxy-6-isoprenyl flavone) is known to be an inhibitor of extermal NADH oxidation by plant mitochondria, while 7-iodo-acridone-4-carboxylic acid (IACA) is an inhibitor of an internal, rotenone-insensitive NAD(P)H dehydrogenase isolated from yeast mitochondria.
Here we show that platanetin inhibits external NAD(P)H oxidation by intact potato ( Solanum tuberosum L. cv. Bintje) tuber mitochondria, deamino-NADH oxidation by Complex I assayed using inside-out submitochondrial particles from these mitochondria, and rotenone-insensitive NAD(P)H oxidation by these submitochondrial particles. IACA was found to inhibit the oxidation of external NADH and succinate by intact mitochondria with similar efficiency. However, IACA also inhibited NADPH and duroquinol oxidation by intact mitochondria as well as deamino-NADH and NAD(P)H oxidation by inside-out submitochondrial particles. This indicates that IACA has several sites of inhibition in the electron transport chain. The lack of specificity of both platanetin and IACA prevents these inhibitors from being used to shed more light on the identity of the NAD(P)H dehydrogenases in plant mitochondria.     

On the relationship between mitochondrial ATPase and the inner membrane

Digestion of the submitochondrial particle (ETP_H) with a proteolytic enzyme, Nagarse, selectively and completely removed the headpieces from the membrane without damaging the electron transfer chain. By determining the amount of protein released by the Nagarse treatment, it was calculated that the headpieces represent 16±0.5% of the total protein of the submitochondrial particles.In respiring ETP_H, membrane-bound AMP was found to be an acceptor of inorganic phosphate, and this esterification led to the formation of membrane-bound ADP. About 70% of the membranebound adenine nucleotides were found to be tightly bound to the intrinsic proteins of the membrane. A transphosphorylation reaction was observed between external and membrane-bound ADP.Abbreviations F₁ coupling factor one - OSCP oligomycin-sensitivity conferring protein - TRU tripartite repeating unit - ETP_H phosphorylating electron transfer particle     

Use of an adenosine triphosphate analog, adenylyl imidodiphosphate, to evaluate adenosine triphosphate-dependent reactions in mitochondria.

Adenylyl imidodiphosphate (AMP-PNP), an analog of adenosine triphosphate (ATP), was found to be an effective inhibitor of adenine nucleotide translocation in rat liver mitochondria. Inhibition by AMP-PNP was shown to be competitive with ATP. Therefore, studies designed to evaluate the interaction of ATP with mitochondrial adenosine triphosphatase (ATPase) in the presence of AMP-PNP were carried out on submitochondrial particles which lack a membrane barrier between the enzyme and the test medium. The effect of AMP-PNP on the ATP-driven reversed electron transfer reaction in sonically prepared submitochondrial particles was further examined by using oligomycin to induce coupling. The ATPase of oligomycin treated particles did not show significantly different sensitivity to AMP-PNP. Submitochondrial particles which were sensitive to AMP-PNP were less efficient in driving energy-coupled reactions. Results from these studies indicate that uncoupling in mitochondria is not only due to a leaky membrane but may also result from an altered membrane-ATPase association.     

Energy-dependent accumulation of the uncoupler picrate and proton flux in submitochondrial particles

In the presence of ATP and oxidizable substrate, submitochondrial particles accumulate up to 7 nmol of picrate/mg of protein. Half of this value is reached at 5 μM picrate in the medium, and maximal energy-dependent accumulation occurs at 25 μM picrate. Mitochondrial proton fluxes calculated under such conditions are 0.80 and 1.08 pmol H⁺/cm²·sec at 10 μM and 25 μM picrate, respectively. These values are similar to those reported for state 4, and are therefore not large enough for uncoupling by picrate through proton translocation. The energy-dependent spectral response of oxonol VI is reversed to 50 % by 40 μM picrate, suggesting that abolishment of membrane potential is responsible for uncoupling of submitochondrial particles by picrate.     

Resolution and Reconstitution of a Mammalian Membrane

The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F ₁, Mg⁺⁺, phospholipids, and F_c. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c ₁, cytochrome c, cytochrome oxidase, phospholipids and Q ₁₀. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin.     

Inhibition of complex I by Ca2+ reduces electron transport activity and the rate of superoxide anion production in cardiac submitochondrial particles

Declines in the rate of mitochondrial electron transport and subsequent increases in the half-life of reduced components of the electron transport chain can stimulate O2*- formation. We have previously shown that, in solubilized cardiac mitochondria, Ca2+ mediates reversible free radical-induced inhibition of complex I. In the study presented here, submitochondrial particles prepared from rat heart were utilized to determine the effects of Ca2+ on specific components of the respiratory chain and on the rates of electron transport and O2*- production. The results indicate that complex I is inactivated when submitochondrial particles are treated with Ca2+. Inactivation was specific to complex I with no alterations in the activities of other electron transport chain complexes. Complex I inactivation by Ca2+ resulted in the reduction of NADH-supported electron transport activity. In contrast to the majority of electron transport chain inhibitors, Ca2+ suppressed the rate of O2*- production. In addition, while inhibition of complex III stimulated O2*- production, Ca2+ reduced the relative rate of O2*- production, consistent with the magnitude of complex I inhibition. Evidence indicates that complex I is the primary source of O2*- released from this preparation of submitochondrial particles. Ca2+ therefore inhibits electron transport upstream of site(s) of free radical production. This may represent a means of limiting O2*- production by a compromised electron transport chain.     

The recognition of two specific binding sites of the adenine nucleotide translocase by palmitoyl CoA in bovine heart mitochondria and submitochondrial particles.

Palmitoyl CoA which is an effective inhibitor of adenine nucleotide transport is able to remove bound [¹⁴C]ADP and [³H]atractylate from the translocator on the outer side of the inner mitochondrial membrane. Bongkrekic acid, when added to the incubation medium prior to palmitoyl CoA, can prevent the removal of bound [¹⁴C]ADP from the membrane by palmitoyl CoA, however, bongkrekic acid is ineffective if palmitoyl CoA is added first. Upon incubation with inverted submitochondrial particles, both palmitoyl CoA and bongkrekic acid prevent the uptake and transport of [¹⁴C]ADP by the particles. Moreover, when the submitochondrial particles are preincubated with [¹⁴C]ADP, palmitoyl CoA, like bongkrekic acid, is unable to remove the bound nucleotide from the inner face of the carrier. Thus, palmitoyl CoA which has a high affinity for the translocator on both sides of the inner mitochondrial membrane, nevertheless, interacts differently with the carrier on each side of the membrane. This suggests that the translocase contains binding sites in two specific states both of which accommodate palmitoyl CoA.     

Inhibition of mitochondrial respiration by analogs of 4-phenylpyridine and 1-methyl-4-phenylpyridinium cation (MPP+), the neurotoxic metabolite of MPTP

In order to clarify the structural requirements associated with the inhibition of mitochondrial respiration by MPP+, the neurotoxic metabolites of the Parkinsonian agent MPTP, ten sets of pyridine/N-methylpyridinium pairs and a few miscellaneous compounds were evaluated on rat liver intact mitochondria (Mw) and on submitochondrial particles (SMP). The pyridinium partners were much more potent inhibitors on Mw than on SMP, indicating that they are concentrated inside mitochondria by the energy-dependent process previously reported for MPP+, probably as a consequence of non-specific passive transport across the mitochondrial inner membrane in response to the transmembrane potential. In the SMP assay, the neutral pyridines were stronger inhibitors than were the pyridinium cations, and the inhibitory potency varied little with structural changes. The N-methylated forms of beta-carbolines may act as endogenous MPP+-like agents.     

Effect of phospholipase A on the structure and functions of membrane vesicles from Mycobacterium phlei.

The phospholipid composition of the electron transport particles and coupling factor-depleted electron transport particles of Mycobacterium phlei are the same, but they differ in contents. The accessibility of partially purified phospholipase A to these membrane phospholipids was found to be different. Treatment of membranes of Mycobacterium phlei with phospholipase A impairs the rate of oxidation as well as phosphorylation. The inhibition of phosphorylation can be reversed by washing the membranes with defatted bovine serum albumin. The reconstitution of membrane-bound coupling factor-latent ATPase activity to phospholipase A-treated depleted electron transport particles and their capacity to couple phosphorylation to oxidation of substrates remained unaffected after phospholipase A treatment. However, the pH gradient as measured by bromthymol blue was not restored after reconstitution of phospholipase A-treated depleted electron transport particles with membrane-bound coupling factor-latent ATPase. These findings show that the phosphorylation coupled to the oxidation of substrates can take place without a pronounced pH gradient in these membrane vesicles. The dye 1-anilino-8-naphthalene sulfonic acid (ANS) exhibited low levels of energized and nonenergized fluorescence in phospholipase A-treated membranes. This decrease in the level of ANS fluorescence in phospholipase A-treated membranes was found to be directly related to the amount of phospholipids cleaved. The decrease in the energy-dependent ANS response in phospholipase A-treated electron transport particles, as compared with untreated electron transport particles, was shown to be a result of a change in the apparent K-d of the dye-membrane complex, and of a decrease in the number of irreversible or slowly reversible binding sites, with no change in the relative quantum efficiency of the dye. The decrease in ANS fluorescence in phospholipase A-treated particles appears to be due to a decrease in the hydrophobicity of the membranes.     

The phosphorylation potential generated by respiring bovine heart submitochondrial particles.

A phosphorylation potential deltaGp, where deltaGp = deltaGo' + RT2.303 log ([ATP]/([ADP][Pi])), of approx. 44.3 kJ.mol-1 (10.6 kcal.mol-1) was generated by submitochondrial particles that were oxidizing either NADH or succinate. Addition of adenylyl imidodiphosphate, which should suppress adenosine triphosphatase activity of any uncoupled particles, did not raise the phosphorylation potential. Raising the Pi concentration slightly increased the magnitude of the value for [ATP]/[ADP], but this did not fully compensate for the increased Pi concentration, so that the phosphorylation potential decreased slightly as the Pi concentration was raised. The phosphorylation potential developed by submitochondrial particles is lower than that generated by phosphorylating membrane vesicles from some bacteria, and is also less than that developed externally by mitochondria, but is strikingly close to the phosphorylation potential that is generated internally by mitochondria.     

Production of reactive oxygen species by mitochondria: central role of complex III

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.     

The ATPase inhibitor protein in oxidative phosphorylation. The rate-limiting factor to phosphorylation in submitochondrial particles.

1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an 'on-line' digital computer. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate ('lag'); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (atp hydrolysis) after all the oxygen has been consumed. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor inhibits both ATP synthesis and hydrolysis by the ATPase. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. The rate constant for inhibitor release was dependent on 'energisation' of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.     

Localization of mitochondrial cytochrome b using specific antibodies]

Specific antibody has been obtained against cytochrome b (pig heart mitochondria). It inhibits the electron transport of the respiratory chain in the intact mitochondria at the cytochrome b site of the inner mitochondrial membrane. It has no effect on the isolated submitochondrial particles which are inside-out inner membrane vescicles free of any outer membrane or outside-out inner membrane. These findings indicate a probably not transmembranous topologic localization of cytochrome b; this component of the respiratory chain seems located near the outer side of the inner mitochondrial membrane.     

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.