Direct action of ethidium bromide upon mitochondrial oxidative phosphorylation and morphology.

Ethidium bromide (23 nmol/mg of protein) was found to be a potent inhibitor of oxidative phosphorylation, as determined by loss of respiratory control through the inhibition of the ADP-induced state-3 rate of oxygen uptake. A time latency for complete loss of respiratory control was noted, after which 2,4-dinitrophenol (DNP) was ineffective in overcoming this inhibition. In the absence of EDTA, ethidium bromide produced an apparent uncoupling, as evidenced by an increase of state-4 rates of oxygen uptake and loss of respiratory control. As low as 8 nmol of ethidium bromide/mg of protein stimulated mitochondrial adenosine triphosphatase (ATPase) for 5 min. Two to three times this amount of ethidium bromide reduced the amount P_i released. Preincubation of mitochondria with ethidium bromide prevented subsequent release of P_i during incubation with ATP. Likewise, preincubation inhibited the DNP-activated ATPase. The uptake of low levels of [¹⁴C]ADP preincubated with ethidium bromide (14 nmol/mg of protein) and succinate or α-ketoglutarate could apparently be reversed, with loss of radioactivity beginning several minutes after addition of the radioactive nucleotide. Inhibition of oxidative phosphorylation by ethidium bromide may be due to modification of the adenine nucleotide transport system in mitochondria. The production of apparently swollen mitochondria treated in vitro with ethidium bromide and substrates necessary for oxidative phosphorylation, as seen in electron micrographs, further indicates that the compound is capable of acting directly upon mouse liver mitochondrial function and structure.     

Phosphate carrier of liver mitochondria: The reaction of its SH groups with mersalyl, 5,5′-dithio-bis-nitrobenzoate,andN-ethylmaleimide and the modulation of reactivity by the energy state of the mitochondria

The inhibitory effect of three SH reagents, mersalyl, 5,5-dithio-bis-nitrobenzoate, andN-ethylmaleimide, on P_i transport in rat liver mitochondria was investigated under a variety of conditions. Mersalyl binds at room temperature with both high (K _d<10 µM) and low affinity to mitochondria. Inhibition of P_i transport by mersalyl goes in parallel with titration of the high-affinity sites, inhibition being complete when 3.5–4.5 nmol/mg protein is bound to the mitochondria. At concentrations of mersalyl equal to or higher than 10 µM, inhibition of P_i transport occurs in less than 10 sec. At concentrations of mersalyl lower than 10 µM, the rate of reaction with the P_i carrier is considerably decreased. At a concentration of 100 µM, 5,5-dithio-bisnitrobenzoate fully inhibits P_i transport in about 1 min at room temperature. Nearly total inhibition is attained when as little as 40–50 pmol/mg is bound to mitochondria. Upon incubation longer than 1 min, additional SH groups, not belonging to the P_i carrier, begin to react. The uncoupler carbonyl cyanidep-trifluoromethoxyphenylhydrazone decreases the rate of reaction of mersalyl, 5,5-dithio-bis-nitrobenzoate, andN-ethylmaleimide with the P_i carrier. Preincubation with P_i has a similar effect. We propose that both carbonyl cyanidep-trifluoromethoxyphenylhydrazone and P_i act by increasing the acidity of the mitochondrial matrix. Protonation of the P_i carrier at the matrix side would change the accessibility of its SH groups at the outer surface of the inner membrane. This might correspond to a membrane-Bohr effect, possibly related to the opening of a gating pore in the P_i carrier.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

Energy-dependent uptake of ochratoxin A by mitochondria.

The interaction of ochratoxin A, a mycotoxin produced by Aspergillus ochraceus, with isolated rat liver mitochondria and plasma membranes has been studied. Cell membranes bind [¹⁴C]ochratoxin A poorly and do not show saturation in the concentration range examined. The uptake of the toxin by mitochondria is saturable, with an apparent K_m at 0 °C of 30 nmol/mg of protein. Sonication or freeze-thawing reduces the extent of incorporation by 88%. Ochratoxin A uptake is energy dependent, resulting in a depletion of intramitochondrial ATP. Uncouplers such as m-chlorocarbonylcyanide phenylhydrazone or the respiratory inhibitors rotenone and antimycin A inhibit uptake 60–85%, while ATP reverses the antimycin and rotenone inhibition. Phosphate transport is sensitive to inhibition by the toxin, as measured by Ca²⁺ plus P_istimulated respiration and [³²P]P_i incorporation. In turn, phosphate inhibits nearly completely [¹⁴C]ochratoxin A uptake at 22 °C and causes a concomitant mitochondrial swelling yet is not incorporated into the matrix space. Thus, the saturable uptake of ochratoxin A is accompanied by a decrease in the energy state and inhibition of P_i transport, which results in deteriorative changes of the mitochondria, as evidenced by large-amplitude swelling.     

Inhibition of anion transport across the mitochondrial membrane by amytal

It has been found that amytal competitively inhibits succinate (+ rotenone) oxidation by intact uncoupled mitochondria. Similar results were obtained in metabolic state 3, the K_i value being 0.45 mM. Amytal did not effect succinate oxidation by broken mitochondria and submitochondrial particles (at a concentration which inhibited succinate oxidation by intact mitochondria). Amytal inhibited the swelling of mitochondria suspended in ammonium succinate or ammonium malate but was without effect on the swelling of mitochondria in ammonium phosphate and potassium phosphate in the presence of valinomycin+carbonylcyanide p-trifluoromethoxyphenylhydrazone.Using [¹⁴C] succinate and [¹⁴C] citrate it has been shown that amytal inhibited the succinate/succinate, succinate/P_i, succinate/malate, and citrate/citrate and citrate/malate exchanges. Amytal inhibited P_i transport across mitochondrial membrane only if preincubated with mitochondria. Other barbiturates: phenobarbital, dial, veronal were found to inhibit [¹⁴C]succinate/anion (P_i, succinate, malonate, malate) exchange reactions in a manner similar to amytal. It is concluded that barbiturates non-specifically inhibit the dicarboxylate carrier system, tricarboxylate carrier and P_i translocator. It is postulated that the inhibition of succinate oxidation by barbiturates is caused mainly by the inhibition of succinate and P_i translocation across the mitochondrial membrane.     

Efflux of magnesium and potassium ions from liver mitochondria induced by inorganic phosphate and by diamide

Addition to rat liver mitochondria of 2 mM inorganic phosphate or 0.15 mM diamide, a thiol-oxidizing agent, induced an efflux of endogenous Mg²⁺ linear with time and dependent on coupled respiration. No net Ca²⁺ release occurred under these conditions, while a concomitant release of K⁺ was observed. Mg²⁺ efflux mediated either by P_i or low concentrations of diamide was completely prevented by EGTA, Ruthenium red, and NEM. These reagents also inhibited the increased rate of state 4 respiration induced both by P_i and diamide. At higher concentrations (0.4 mM), diamide induced an efflux of Mg²⁺ which was associated also with a release of endogenous Ca²⁺. Under these conditions EGTA completely prevented Mg²⁺ and K⁺ effluxes, while they were only partially inhibited by Ruthenium red and NEM. It is assumed that Mg²⁺ efflux, occurring at low diamide concentrations or in the presence of phosphate, is dependent on a cyclic in-and-out movement of Ca²⁺ across the inner mitochondrial membrane, in which the passive efflux is compensated by a continuous energy linked reuptake. This explains the dependence of Mg²⁺ efflux on coupled respiration, as well as the increased rate of state 4 respiration. The dependence of Mg²⁺ efflux on phosphate transport is explained by the phosphate requirement for Ca²⁺ movement.Abbreviations Diamide diazenedicarboxylic acidbis-dimethylamide - FCCP p-trifluoromethoxyphenylhydrazone - EGTA ethylene glycol-bis-(2-amino ethyl ether)-N,N-tetracetic acid - P_i inorganic phosphate - Ruthenium red Ru₂(OH)₂Cl₄ · 7NH₃ · 3H₂O - state 4 controlled state of respiration in the presence of substrate - RCI respiratory control index - NEM N-ethyl maleimide A partial and preliminary report of these results has been published inBiochem. Biophys. Res. Comm.,78 (1977) 23.     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

Characterization of orthophosphate-induced active cation transport in isolated liver mitochondria

The kinetics of the P_i-induced active transport of ions by isolated liver mitochondria were studied by monitoring photometrically mitochondrial volume changes. In a previous communication, these volume changes were shown to correlate quantitatively with the net uptake or release of ions. In the present study the specificity of the P_i role was further characterized. The data support the contention that cations are actively transported. Anions follow the transfer of cations to maintain electrical neutrality. The relationship of the transport system to oxidative phosphorylation was investigated by simultaneously monitoring both processes under different experimental conditions. The results of the experiments are quantitatively consistent with a model proposed for the P_i-induced active transport in isolated rat liver mitochondria. The model includes the following features. 1. P_i induces an inwardly directed, carrier-mediated active transport of cations. 2. The transport is coupled to the energy-conserving reactions of the cytochrome chain. 3. The efflux of ions accumulated in the presence of low P_i concentrations occurs by passive diffusion. 4. Net accumulation ceases when the rates of active transport and passive diffusion become equal. 5. The active transport competes with oxidative phosphorylation for a common, nonphosphorylated, high-energy intermediate.     

Swelling and contraction of heart mitochondria suspended in ammonium phosphate

Bovine heart mitochondria which have been allowed to swell in isotonic NH ₄ ⁺ phosphate contract in response to initiation of oxidative phosphorylation. The contraction occurs optimally at pH 6.0 and appears from inhibition studies to result from P_i uptake being slower than removal of internal P_i via phosphorylation of external ADP. Similar results are obtained when K⁺ + nigericin is substituted for NH ₄ ⁺ . Mersalyl inhibition of P_i transport in respiring, nonphosphorylating mitochondria which have been allowed to swell in NH ₄ ⁺ phosphate reveals a contractile process having an alkaline pH optimum. This contraction resembles closely the contraction observed in salts of strong acids and presumably occurs by electrophoretic ejection of P_i anions driven by electrogenic H⁺ ejection.     

Ca2+ Induces a Cyclosporin A-Insensitive Permeability Transition Pore in Isolated Potato Tuber Mitochondria Mediated by Reactive Oxygen Species

Oxidative damage of mammalian mitochondria induced by Ca²⁺ and prooxidants is mediated by the attack of mitochondria-generated reactive oxygen species on membrane protein thiols promoting oxidation and cross-linkage that leads to the opening of the mitochondrial permeability transition pore (Castilho et al., 1995). In this study, we present evidence that deenergized potato tuber (Solanum tuberosum) mitochondria, which do not possess a Ca²⁺ uniport, undergo inner membrane permeabilization when treated with Ca²⁺ (>0.2 mM), as indicated by mitochondrial swelling. Similar to rat liver mitochondria, this permeabilization is enhanced by diamide, a thiol oxidant that creates a condition of oxidative stress by oxidizing pyridine nucleotides. This is inhibited by the antioxidants catalase and dithiothreitol. Potato mitochondrial membrane permeabilization is not inhibited by ADP, cyclosporin A, and ruthenium red, and is partially inhibited by Mg²⁺ and acidic pH, well known inhibitors of the mammalian mitochondrial permeability transition. The lack of inhibition of potato mitochondrial permeabilization by cyclosporin A is in contrast to the inhibition of the peptidylprolyl cis–trans isomerase activity, that is related to the cyclosporin A-binding protein cyclophilin. Interestingly, the monofunctional thiol reagent mersalyl induces an extensive cyclosporin A-insensitive potato mitochondrial swelling, even in the presence of lower Ca²⁺ concentrations (>0.01 mM). In conclusion, we have identified a cyclosporin A-insensitive permeability transition pore in isolated potato mitochondria that is induced by reactive oxygen species.     

Phosphate transport in rat liver mitochondria--a mobile carrier model.

The possibility of a mobile carrier model for phosphate transport in rat liver mitochondria was examined on the basis of counterflux experiments. The rate of P_i uptake and P_i exchange were identical and depended on the external pH value. The alkalization of the suspending medium of P_i-preloaded mitochondria induced an efflux of P_i. The induction of a net P_i efflux by alkalization of the external medium stimulated the rate of ³²P_i uptake. Arsenate was shown to be an alternative substrate for P_i-carrier. A net efflux of inorganic arsenate (As_i), induced by alkalization of the external medium, also supported an acceleration of the ³²P_i uptake. When mitochondria were first preloaded with As_i and ³²P_i and then diluted into a more alkaline buffer free of Asi, a transient uptake of ³²P_i was observed. These results are discussed in terms of reorientation of the active site of the P_i carrier under the conditions where a net efflux of P_i or As_i occurred. This conclusion was supported by a change in the accessibility of the SH groups of the carrier toward poorly permeant thiol reagents during that process.     

Phosphate transport in rat liver mitochondria: Location of sulfhydryl groups essential for transport activities

The membrane orientation and symmetry of protein thiol group(s) necessary for transport of P_i in rat liver mitochondria have been assessed by comparing inhibition of transport in intact mitochondria to that in inverted vesicles of purified inner membrane. The permeability characteristics of a variety of inhibitors have been determined under specified conditions. The sensitivities of the uptake pathways in mitochondria and in inverted vesicles appear thus far to be identical. By comparing results with permeant and nonpermeant inhibitors, or sequential treatment with different inhibitors, arguments can be made in favor of a single reorienting site of thiol sensitivity.DABS p-(diazonium)-benzenesulfonic acid - IMV inner membrane vesicles - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - GSH reduced glutathione - TMPD N,N,NN-tetramethyl-p-phenylenediamine - EGTA ethylene glycol-bis (-aminoethyl ether) - p-CMB p-chloromercuribenzoate - NEM N-ethylmaleimide - FCCP p-trifluoromethoxycarbonyl cyanide phenylhydrazone     

Tetradifon: an oligomycin-like inhibitor of energy-linked activities of rat liver mitochondria

Tetradifon (p-chlorophenyl-2,4,5-trichlorophenyl sulfone) at concentrations between 4.5 and 27.0 nmoles/mg mitochondrial protein provides half-maximal inhibition of the following energy-linked activities of rat liver mitochondria: ADP-stimulated respiration, DNP-stimulated ATPase activity, Mg⁺⁺-stimulated ATPase activity, and P_i-ATP exchange activity. Tetradifon has no effect on the activity of soluble ATPase purified from rat liver mitochondria. Respiration inhibited by tetradifon is restored upon addition of 2,4-dinitrophenol. It is concluded that tetradifon acts at or near the oligomycin sensitivity conferring complex located in the mitochondrial inner membrane.     

Effects of ranolazine on fatty acid transformation in the isolated perfused rat liver

It has been proposed that in the heart, ranolazine shifts the energy source from fatty acids to glucose oxidation by inhibiting fatty acid oxidation. Up to now no mechanism for this inhibition has been proposed. The purpose of this study was to investigate if ranolazine also affects hepatic fatty acid oxidation, with especial emphasis on cell membrane permeation based on the observations that the compound interacts with biological membranes. The isolated perfused rat liver was used, and [1-¹⁴C]oleate transport was measured by means of the multiple-indicator dilution technique. Ranolazine inhibited net uptake of [1-¹⁴C]-oleate by impairing transport of this fatty acid. The compound also diminished the extra oxygen consumption and ketogenesis driven by oleate and the mitochondrial NADH/NAD⁺ ratio, but stimulated ¹⁴CO₂ production. These effects were already significant at 20 μM ranolazine. Ranolazine also inhibited both oxygen consumption and ketogenesis driven by endogenous fatty acids in substrate-free perfused livers. In isolated mitochondria ranolazine inhibited acyl-CoA oxidation and β-hydroxybutyrate or α-ketoglutarate oxidation coupled to ADP phosphorylation. It was concluded that ranolazine inhibits fatty acid uptake and oxidation in the liver by at least two mechanisms: inhibition of cell membrane permeation and by an inhibition of the mitochondrial electron transfer via pyridine nucleotides.     

Effects of Ipomeamarone on Respiratory Enzyme System in Mitochondria

Ipomeamarone inhibited oxidation and phosphorylation in tightly coupled rat liver mitochondria. The inhibition of the oxygen uptake was higher when either β-hydroxybutyrate or α-ketoglutarate was supplied as the substrate than when succinate was used. In mitochondrial preparations which had been uncoupled, inhibitions of the electron transport chain from β-hydroxybutyrate to cytochrome c, and of the enzymes succinate cytochrome c oxidoreductase and β-hydroxybutyrate dehydrogenase were observed. Ipomeamarone inhibited also the ATP-inorganic phosphate exchange reaction, but did not act as an uncoupler; it repressed 2, 4-dinitropheaol-induced oxygen uptake.     

The pathway of inorganic-phosphate efflux from isolated liver mitochondria during adenosine triphosphate hydrolysis

1. The distribution of P_i between mitochondria and suspending medium during uncoupler-stimulated hydrolysis of ATP by rat liver mitochondria [Tyler (1969) Biochem. J. 111, 665–678] has been reinvestigated, by using either mersalyl or N-ethylmaleimide as inhibitors of P_i transport and either buffered sucrose/EDTA or LiCl/EGTA solutions as suspending medium. More than 75% of the total P_i liberated was retained in mitochondria treated with either inhibitor at all ATP concentrations tested (0.2–2.5mm). With low ATP concentrations and mersalyl-treated mitochondria incubated in sucrose/EDTA, virtually all the P_i liberated was retained in the mitochondria. 2. Larger amounts of P_i appeared in the suspending medium during ATPase activity, despite the presence of N-ethylmaleimide, when LiCl/EGTA was used as suspending medium compared with sucrose/EDTA. Two sources of this P_i were identified: (a) a slow efflux of P_i from mitochondria to suspending medium despite the presence of N-ethylmaleimide; (b) a slow ATPase activity insensitive to carboxyatractyloside, which was stimulated by added Mg²⁺, partially inhibited by oligomycin or efrapeptin and strongly inhibited by EDTA. 3. It is concluded that liver mitochondria preparations contain two distinct forms of ATPase activity. The major activity is associated with coupled mitochondria of controlled permeability to adenine nucleotides and P_i and is stimulated strongly by uncoupling agents. The minor activity is associated with mitochondria freely permeable to adenine nucleotides and P_i, is unaffected by uncoupling agents and is activated by endogenous or added Mg²⁺. 4. When mitochondria treated with mersalyl were incubated in buffered sucrose solution, almost all the P_i liberated was recovered in the suspending medium, unless inhibitors of P_i-induced large-amplitude swelling such as EDTA, EGTA, antimycin, rotenone, nupercaine or Mg²⁺ were added. Thus the loss of the specific permeability properties of the mitochondrial inner membrane associated with large-amplitude swelling also influences the extent of P_i retention during ATPase activity. 5. The results confirm the previous conclusion (Tyler, 1969) that the P_i transporter provides the sole pathway for P_i efflux during uncoupler-stimulated ATP hydrolysis by mitochondria. It is concluded that more recent hypotheses concerning the influence of Mg²⁺ on mersalyl inhibition of the P_i transporter [Siliprandi, Toninello, Zoccaroto & Bindoli (1975) FEBS Lett. 51, 15–17] and a postulated role of the adenine nucleotide exchange carrier in P_i efflux [Reynafarje & Lehninger (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4788–4792] are erroneous and should be discarded.     

In vitro studies of p32 uptake in mouse liver mitochondria

Isolated mouse liver mitochondria were incubated in two types of P³²-labelled sucrose-phosphate buffers. The first contained no added ATP or oxidizable substrate. The second contained added ATP. Samples were taken at specified times, up to 60 minutes, and analyses were made of the mitochondrial TCA-soluble inorganic P³² and the total mitochondrial residue P³¹ and P³². The results of the analyses showed that when the phosphorus inhibition index (the ratio of the amount of incubation inorganic phosphorus to the square of the amount of tyrosine in the mitochondria) was high, inorganic P³² uptake was low and vice versa. In accordance with established data, increased P³² uptake was obtained when ATP was added. ATP was found to stabilize the turnover of TCA-insoluble residue phosphorus as well as to maintain the TCA-soluble orthophosphate pool. These results support findings regarding the inhibitory and controlling effects of incubation medium phosphate in the regulation of inorganic phosphorus uptake.     

Stimulation of septation in mitochondria by diamide,a thiol oxidising agent

Summary Diamide (10^–4M), a thiol oxidizing agent, rapidly promotes septation in isolated frog liver mitochondria and also in situ in liver slices. The effect is partially inhibited by dithioerythritol. DNP does not have this effect, and it is concluded that diamide does not promote septation via an uncoupling action. The septate mitochondria have a different appearance from typical dividing mitochondria previously described; it is suggested that (1) diamide may act by favouring the fusion of the internal membranes, and (2) -SH oxidation is of importance in mitochondria in ageing and in various pathological conditions. The ways in which septa may develop in mitochondria in the orthodox and condensed configuration are discussed.     

Phosphate transport in mitochondria: Past accomplishments,present problems,and future challenges

The requirement of inorganic phosphate (P_i) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific P_i transport systems. The mitochondrial proton/phosphate symporter (P_ic) is a membrane-embedded protein which translocates P_i from the cytosol into the mitochondrial matrix. P_ic is responsible for the very rapid transport of most of the P_i used in ATP synthesis. During the past five years there have been advances on several fronts. Genomic and cDNA clones for yeast, bovine, rat, and human P_ic have been isolated and sequenced. Functional expression of yeast P_ic in yeast strains deficient in P_i transport and expression inEscherichia coli of a chimera protein involving P_ic and ATP synthase subunit have been accomplished. P_ic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria. Six transmembrane segments appear to be a structural feature shared between P_ic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin. These recent advances on P_ic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/