Effects of fluorescamine labeling on mitochondrial ATPase activity.

Fluorescamine labeling of rat liver mitochondria enhances the ATPase activity. It reached maximum stimulation when mitochondria were treated with 30–34 nmol fluorescamine per mg of mitochondrial protein. This stimulation is inhibited by N,N′-dicyclohexylcarbodiimide. The maximum stimulation caused by labeling is the same as that obtained from uncoupler with optimum concentration. The chemiosmotic potential (Δμ_H⁺) decreases as the labeling increased. However, Δμ_H⁺ is not abolished completely even when ATPase activity reaches a maximum. The results suggest that primary amino groups may be involved in controlling mitochondrial ATPase activity.     

Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

Isolation and properties of OSCP and an F1-ATPase binding protein from rat liver mitochondria - evidence against OSCP as the linking "stalk" between F1 and the membrane.

Oligomycin Sensitivity Conferral Protein (OSCP) and an F₁-ATPase Binding Protein were isolated from F₁-depleted rat liver mitochondrial membrane. Their molecular weights on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea were 22,500 and 8,500 respectively. When incubated with liver TUA (trypsin, urea and ammonia-treated) submitochondrial particles, the binding protein was effective in the binding of F₁ to the particles with the resultant particle-bound ATPase activity not oligomycin sensitive. When OSCP was then incubated with the reconstituted membrane-bound ATPase, its activity became oligomycin sensitive. These results suggest that, first; the binding protein, but not OSCP, connects F₁-ATPase to the membrane of rat liver mitochondria and maybe to the “stalk”, if indeed there is a stalk in mitochondrial membrane ATPase complex; and second; the function of OSCP is solely to render the ATPase activity sensitive to oligomycin and other similar inhibitors.     

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

Properties of the ATPase activity associated with peroxisome-enriched fractions from rat liver: comparison with mitochondrial F1F0-ATPase

Highly purified peroxisomal fractions from rat liver contain ATPase activity (18.8 ± 0.1 nmol/min per mg, n = 6). This activity is about 2% of that found in purified mitochondrial fractions. Measurement of marker enzyme activities and immunoblotting of the peroxisomal fraction with an antiserum raised against the β-subunit of mitochondrial ATPase indicates that the ATPase activity in the peroxisomal fractions can not be ascribed to contamination with mitochondria or other subcellular organelles. From the sensitivity of the ATPase present in the peroxisomal fraction towards a variety of ATPase inhibitors, we conclude that it displays both V-type and F-type features and is distinguishable from both the mitochondrial F₁F₀-ATPase and the lysosomal V-type ATPase.     

Short-term vitamin A supplementation at therapeutic doses induces a pro-oxidative state in the hepatic environment and facilitates calcium-ion-induced oxidative stress in rat liver mitochondria independently from permeability transition pore formation

There is a growing body of evidence showing that vitamin A induces toxic effects in several experimental models and in human beings. In the present work, we have investigated the effects of short-term vitamin A supplementation on the adult rat liver redox status. We have found that vitamin A at therapeutic doses induces a hepatic oxidative insult. Furthermore, we have observed increased antioxidant enzyme activity in the liver of vitamin-A-treated rats. Additionally, some mitochondrial dysfunction was found since superoxide anion production was increased in vitamin-A-treated rat liver submitochondrial particles, which may be the result of impaired mitochondrial electron transfer chain activity, as assessed here. We have also isolated rat liver mitochondria and challenged it with 75 μM CaCl₂, a non-oxidant agent that is able to induce mitochondrial oxidative stress indirectly. We have found that mitochondria isolated from vitamin-A-treated rat liver are more sensitive to CaCl₂ than control mitochondria regarding the redox status. Importantly, vitamin A seems to alter mitochondrial redox status independently of the participation of the mitochondrial permeability transition pore, which is activated by Ca²⁺ ions since cyclosporin A did not prevent the oxidative insult elicited by Ca²⁺ addition. Overall, we show here that mitochondria are a target of vitamin-A-associated toxicity also in vivo.     

Adenosine triphosphatase from rat liver mitochondria: separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.

Homogeneous ATPase from rat liver mitochondria binds one mole of ADP per mole of enzyme reversibly, and with high affinity (K_D = 1–2 μM). The high affinity binding site is highly specific for ADP and dADP. AMP does not bind. Agents which inhibit ATP hydrolysis have little inhibitory effect on the high affinity binding of ADP. These agents include adenylyl imidodiphosphate (AMP-PNP), azide, sucrose, and the divalent cation Mg⁺⁺. AMP-PNP inhibits ATPase activity in phosphorylating membrane preparations of rat liver mitochondria by about 90 percent, but is without effect on ATP synthesis. These results are consistent with the view that the purified soluble, and the membrane-bound ATPase of rat liver mitochondria contain separate sites involved in ATP hydrolysis and in the reversible, high affinity binding of ADP.     

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.     

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.     

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/     

The antifertility agent,gossypol, changes several mitochondrial functions in the presence of Mg2+

1. The effect of gossypol in the presence of K⁺ or Mg²⁺, or both, was studied on ATPase activity and respiration of rat liver mitochondria.2. Respiration was uncoupled in the presence of gossypol, Mg²⁺, and K⁺, whereas in the presence of gossypol and Mg²⁺ a partial inhibition was observed.3. Gossypol stimulated ATPase activity in the presence of K⁺ or Mg²⁺, but maximal activity was observed when both cations were in the incubation medium.4. Stimulation of ATPase activity in the presence of Mg²⁺ was dose related.5. EDTA reverted the stimulation produced by gossypol on ATPase activity.6. Gossypol had no effect on the ATPase activity of submitochondrial particles, which suggests an indirect action of gossypol on the enzyme.7. Mitochondrial membrane potential showed a higher collapse in the presence of gossypol and 1mM MgCl₂.8. The observed effects of gossypol could be explained by the collapse of the mitochondrial membrane potential.     

Effect of Sclerin on Incorporation of Oleic Acid into Phospholipids and Activity of Phospholipase in Mitochondria

Sclerin (SCL) stimulated the oxidation and the incorporation into the phospholipids of Na-[1-¹⁴C]-oleate in mitochondria isolated from rat liver, preventing the depression of the phosphorylating functions and protecting 2,4-dinitrophenol (DNP)-activated ATPase in mitochondria during incubation with oleate. Also, SCL markedly enhanced the activity of phospholipase to hydrolyze endogenous substrates in mitochondria. The increase in the activity was due to reconstruction of phospholipids through esterification of oleate in mitochondrial membrane, but not to the de novo enzyme synthesis. It was concluded that the level of endogenous phospholipase in mitochondria during incubation reflects the energy- dependent reacylation of the lysophospholipids produced by the action of phospholipase in mitochondrial membrane.     

Differential effects of 2,4-dinitrophenol and valinomycin (+K+) on uncoupler-stimulated ATPase of human tumor mitochondria

The uncoupler-stimulated mitochondrial ATPase of four human tumors, mouse kidney, brain and fetal liver exhibited a characteristic behavior when preincubated with the H⁺-conducting uncouplers, dinitrophenol, CCCP, S-13 and gramicidin. The ATPase activity was considerably lower with preincubation than without. Preincubation with valinomycin (+K⁺), on the other hand, did not result in a significant decrease of the ATPase activity. These results may be contrasted with those obtained with liver or heart mitochondria, the ATPase activity of which did not suffer any loss when preincubated with dinitrophenol. The effect of preincubation with dinitrophenol on the tumor mitochondria could not be accounted for by dinitrophenol-induced Mg²⁺ efflux, since the differential effects of dinitrophenol and valinomycin (+K⁺) remained even when ATPase activity was determined in presence of Mg²⁺. Small amounts of ATP and ADP in the preincubation mixture containing dinitrophenol protected against the decay of the ATPase activity, implicating the exchangeable adenine nucleotides in the tumor mitochondria. In a model system where liver mitochondria were depleted of their adenine nucleotides, a lower ATPase activity was indeed obtained. However, direct determination of the concentations of adenine nucleotides in dinitrophenol- and valinomycin-treated tumor mitochondria revealed only slight differences.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al., 1980, Biochem. Biophys. Res. Commun.92, 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg²⁺ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg²⁺ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg²⁺ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg²⁺ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg²⁺ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg²⁺ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg²⁺ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.     

Inhibition of mitochondrial ATPase by 2,4,3',5'-tetrahydroxystilbene.

2,4,3',5'-tetrahydroxystilbene (THS) wa s found to inhibit rat liver mitochondrial adenosine triphosphatase (ATPase) activity induced by various concentrations of 2,4-dinitrophenol (DNP). The I50 was found to be 17 nmoles/mg mitochondrial protein. The maximum inhibitory effects of oligomycin and atractyloside on the DNP-activated mitochondrial ATPase activity can be enhanced by adding THS. The atractyloside-insensitive ATPase activity of Lubrol-treated rat liver mitochondria was also inhibited by low concentration of THS. The tetramethoxyderivative of THS was much less effective than the parent compound in depressing the ATPase activity of both intact and Lubrol-treated mitochondria. These observations suggest that the phenolic groups are essential for the mitochondrial actions of THS, and this compound most probably acts by a mechanism different from oligomycin on the mitochondrial ATPase complex.     

A possible mechanism for the increased oxidation of choline after chronic ethanol ingestion

An attempt has been made to determine the location of the site at which the metabolism of ethanol interacts with that of choline to produce an increase in the oxidation of choline. The first enzyme in the oxidation pathway for choline, choline dehydrogenase, was assayed using a newly developed spectro-photometric assay and freshly isolated intact rat liver mitochondria. No changes were observed in either the ‘apparent’ V or the ‘apparent’ K_m values of choline dehydrogenase for choline after ethanol ingestion. However, when the choline oxidase system was assayed, a 28% decrease in ‘apparent’ K_m for choline and a 53% increase in ‘apparent’ V was observed. The effects of ATP on choline oxidase were studied further, and a 29.4% decrease was observed in mitochondrial ATP levels from freshly isolated mitochondria from the ethanoltreated rats. In vitro aging of mitochondria further decreased the level of ATP, and the rate of decrease was considerably faster during the first hour in the mitochondria from the ethanol-treated animals. The decreases in ATP from both control and experimental mitochondria were accompanied by increases in choline oxidase activity. The initial decrease in ATP was correlated with an increase in mitochondrial ATPase activity which may be related to an increase in mitochondrial Mg²⁺. Because chronic ethanol ingestion has resulted in decreased oxidation rates of succinate and β-hydroxybutyrate while at the same time increasing the oxidation rates of choline, the studies reported here suggest that the effect of chronic ethanol ingestion is primarily on a step that is unique to choline and which probably exists prior to the electron transport chain.     

Content and binding characteristics of the mitochondrial ATPase inhibitor,IF1 in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms

We determined the IF₁ contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF₁-mediated inhibition of the ATPase activity of IF₁-depleted rabbit heart mitochondrial particles (RHMP) by IF₁-containing extracts from these same eight species. The IF₁ binding experiments were run at both species-endogenous IF₁ levels and at an IF₁ level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF₁ or either speciesendogenous or normalized levels of pig heart IF₁ were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF₁-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF₁ were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF₁-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF₁ inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF₁ inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF₁, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF₁, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF₁. In contrast, rat and mouse heart mitochondria contain only low levels of IF₁ which exhibit binding characteristics similar to those of the pig and rabbit heart inhibitor. The guinea pig is the only mammal thus far examined that contains a loweraffinity form of IF₁. In the present study we also determined the IF₁ contents and IF₁-to-F₁ ATPase activity ratios of cardiac muscle, skeletal muscle, liver, and brain mitochondria of rabbit, pigeon, and rat, species representative of the three homeothermic regulatory classes.     

A method for isolating lung mitochondria from rabbits, rats, and mice with improved respiratory characteristics.

Previous methods for isolating lung mitochondria, particularly from rabbits, have yielded preparations which exhibit low respiratory control ratios (RCRs). We now report a method for the isolation of lung mitochondria from rabbit, rat, and mouse with RCRs, ADP/O ratios, and rates of substrate oxidation comparable to those for liver mitochondria. These mitochondrial preparations fail to oxidize exogenously added NADH and exhibit RCRs, during succinate oxidation, which closely approximate those obtained with NADH-linked substrates. However, an otherwise latent Mg²⁺-stimulated ATPase activity can still be elicited when Mg²⁺ is added to the mitochondrial incubation medium. This ATPase activity is insensitive to oligomycin and atractyloside, indicating that the source is from contaminating endoplasmic reticulum. The pH and EDTA concentration for maximum substrate oxidation and RCR were found to be 7.2 and 0.1 mm, respectively. State 4 respiration was affected by pH and EDTA concentration while state 3 respiration appeared to be independent of these two factors over the ranges studied.     

Divalent cation chelators citrate and EDTA unmask an intrinsic uncoupling pathway in isolated mitochondria

We demonstrate a suppression of ROS production and uncoupling of mitochondria by exogenous citrate in Mg²⁺ free medium. Exogenous citrate suppressed H₂O₂ emission and depolarized mitochondria. The depolarization was paralleled by the stimulation of respiration of mitochondria. The uncoupling action of citrate was independent of the presence of sodium, potassium, or chlorine ions, and it was not mediated by the changes in permeability of the inner mitochondrial membrane to solutes. The citrate transporter was not involved in the citrate effect. Inhibitory analysis data indicated that several well described mitochondria carriers and channels (ATPase, IMAC, ADP/ATP translocase, mPTP, mKATP) were not involved in citrate’s effect. Exogenous MgCl₂ strongly inhibited citrate-induced depolarization. The uncoupling effect of citrate was demonstrated in rat brain, mouse brain, mouse liver, and human melanoma cells mitochondria. We interpreted the data as an evidence to the existence of a hitherto undescribed putative inner mitochondrial membrane channel that is regulated by extramitochondrial Mg²⁺ or other divalent cations.