DNP-stimulated ATPase Activity Research Articles

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 by Fusarium 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 Ca(2+)-uptake of mitochondria energized both by ATP or succinate without affecting the Ca(2+)-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the Pi 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.

EFFECTS OF DEMETHYLCHLORTETRACYCLINE PHOTOTOXICITY ON THE STRUCTURE and FUNCTIONS OF RAT LIVER MITOCHONDRIA

The phototoxic effects of demethylchlortetracycline (DMCT) with UVA radiation on isolated rat liver mitochondria were studied. DMCT at concentration of 21.5 microM with 5.53 x 10(-2) J cm-3 of UVA was found to be a potent uncoupler of oxidative phosphorylation. DMCT alone also uncoupled mitochondria but at higher concentrations (105 microM). ATPase activity was remarkably induced in mitochondria exposed to DMCT (21.5 microM) plus UVA (120% of DNP-stimulated ATPase activity). Content of ATP in such mitochondria when measured after addition of ADP was much smaller than that in control mitochondria. Ultrastructurally, mitochondria treated either with DMCT (21.5 microM) or with UVA alone stayed in the condensed configuration. On the other hand, uncoupled mitochondria treated with DMCT plus UVA became swollen and were changed into the orthodox configuration.

Ｉｓｏｆｌｏｘｙｔｈｅｐｉｎのラット肝ミトコンドリア膜に対する作用

The effect of isofloxythepin (IFT) on isolated rat liver mitochondria was examined.In the presence of β-hydroxybutyric acid, succinic acid or ascorbic acid as respiratory substrate, IFT accelerated the state 4 respiration with a dose-dependent manner at low concentrations (≤100 μM), but did not show any noticeable influence on the state 3 respiration.IFT stimulated the latent ATPase activity at low concentrations. But at higher concentrations, the DNP-stimulated ATPase activity was decreased remarkably. The activities of the latent and DNP-stimulated ATPases were at the same level when IFT at a concentration of 400uM was added. These results suggest that IFT influences isolated rat liver mitochondrial functions as an uncoupler.

Impairment of mitochondrial respiratory activity in the early ischemic myocardium--with special reference to electron transport system.

Impairment of mitochondrial respiration in early myocardial ischemia was studied with special reference to myocellular irreversible injury. The technique used was total ligation of the left anterior descending coronary artery, followed by reconstruction of coronary blood flow, in the dog. State 3 respiratory activity reduced significantly to 76% of that of the non-ischemic myocardium in subendocardial muscle (Endo) as early as 30 min after occlusion, and at 60 min to 84% in the subepicardium (Epi). The activity was not recovered by reperfusion. The activity of complex I of sonicated submitochondrial particles decreased at 30 min to 67% in Endo and at 60 min to 71% in Epi, and was not recovered by reperfusion. Complex II and IV activities were kept in the control level until 60 min of ischemia. DNP-stimulated ATPase activity reduced to 79% in Endo at 15 min and to 70% in Epi at 30 min, but recovered significantly by reperfusion until 30 min of ischemia. Mitochondrial respiratory activity was impaired irreversibly in ischemia for 30 min in Endo and this spread to Epi later. Degradation of complex I is considered to be one of the causes of myocardial irreversibility in early ischemia.

The functional and ultrastructural changes of hepatic mitochondria in acute experimental pancreatitis in dogs treated with prostacyclin

Acute pancreatitis affects the subcellular status of the liver both by enzymatic toxemia and by the impairment of the protective function exerted by a normal pancreas on the liver. The aim of this study was to investigate the effect of the cytoprotective agent prostacyclin (PGI 2 ) on hepatic mitochondria during acute experimental pancreatitis (AEP) in dogs. AEP was induced by the injection of a bile and trypsin mixture into the pancreatic duct, and experiments were terminated after 12 hrs with the exception of dogs with AEP 24 hrs (N = 5). The hepatic mitochondria from the group with AEP 12 hrs without treatment (N = 5) showed a significant impairment of succinate oxidation in St. 3 (with ADP) to 20.1 nanoatoms O×min −1 × mg −1 of mitochondrial protein in comparison to healthy dogs (N = 4) −37.6 units. The respiratory control ratio (RCR) in the former group was half as great as in the control group (1.56 and 3.13 respectively). The ADP: 0 ratio in this group was impossible to calculate. DNP-stimulated ATP-ase activity was lowered to 50.6 nM Pi min −1 mg −1 of protein in comparison to the healthy animals (642.7 units). In dogs AEP 12 hrs treated with PGI 2 in the dose of 20 ng/kg · min for 12 hrs (N = 5) and in the group additionally pretreated with PGI 2 for 1 hr before AEP (N = 5), the respective values of succinate oxidation in St. 3 were 22.2 and 26.5 units; RCR with ADP 2.05 and 2.07; ADP: O ratio 1.10 and 1.19 (in healthy dogs ADP: O = 1.90). DNP-stimulated ATP-ase activity was also augmented in comparison with the untreated group, 94.0 and 125.6 units, respectively. In the group with AEP 12 hrs without treatment, the ultrastructural examination of the liver showed severely damaged, mitochondria with swelling, destruction of limiting membranes and cristae. In the group with AEP 24 hrs without treatment the functional and morphological picture of mitochondria was improved in comparison to AEP 12 hrs and was comparable to treated groups. Prostacyclin in investigated dosage exerts a protective effect on hepatic mitochondria, damaged during acute experimental pancreatitis in dogs.

Interaction of oleficin with the inner membrane of rat liver mitochondria.

The effects of oleficin, a polyene antibiotic of the nonmacrolide type, on isolated rat liver mitochondria were studied. Oleficin at a concentration of about 10 nmoles/mg protein increases both the rate of state 4 respiration and the "basal" ATPase activity of mitochondria. In contrast to this it inhibits the rate of both state 3 and uncoupled respiration and the DNP-stimulated ATPase activity. These inhibitions can be prevented by low concentrations (2 approximately 5 mM) of magnesium ions. Oleficin induces a high amplitude swelling of non-respiring mitochondria in the isoosmotic nitrate and chloride solutions of K+, Na+, Tris+, Tea+ or Mg2+. In contrast to that it does not induce swelling of mitochondria treated with ruthenium red in isoosmotic calcium acetate. Indirect evidence suggests that oleficin increases also the proton permeability of the inner membrane. The swelling observed in the isoosmotic solutions of monovalent cations can be prevented by low concentration (2 approximately 5 mM) of Mg2+. In the presence of the antibiotic Mg2+ and Ca2+ but not K+ and Na+, are transferred from an aqueous phase into a butanol-toluene bulk phase. Oleficin depletes Mg2+ and Ca2+ from mitochondria in a concentration dependent manner. Complete depletion of Mg2+ occurs only in the presence of EDTA, while that of Ca2+ does not need the chelator. It is concluded that the effects of oleficin on mitochondrial functions can be explained on the basis of an increase of the inner membrane permeability as the consequence of the depletion of Mg2+ from mitochondria caused by the antibiotic.

Thiols related to mitochondrial ATPase and transports: Unmasking upon conformational changes supported by the comparative effects of ethacrynate and dihydroethacrynate

Comparison between the effects on various rat liver mitochondrial functions of ethacrynate, a thiol reagent inhibitor of oxidative phosphorylations [3, 4] and those of dihydroethacrynate its saturated derivative which is not a thiol reagent, has been performed. Both, ethacrynate and dihydroethacrynate increase oxygen consumption by mitochondria in state 4 (succinate as substrate) in a concentration dependent way (from 1 to 5 X 10(-4) M EA or DHEA). This activation is followed, only with ethacrynate, by an inhibition appearing sooner with higher concentrations. After preincubation or mitochondria with ethacrynate (1 to 5 X 10(-4) M), the stimulation of respiration by (ADP + Pi) is completely inhibited whereas it is only weakly affected by dihydroethacrynate at the same concentrations. Ethacrynate and dihydroethacrynate provoke variations of intramitochondrial Mg2+ and K+ levels which need energy from the respiratory chain. These are affected by Pi or (Pi + ADP) in a different way with ethacrynate and with dihydroethacrynate. After preincubation with mitochondria, ethacrynate and to a smaller extent dihydroethacrynate, inhibit partially ADP translocation; ADP increases the inhibitory effect of EA on translocation and not that of dihydroethacrynate. Ethacrynate increases the oligomycin sensitive ATPase activity and dihydroethacrynate still more. After a ten minutes preincubation with mitochondria, ethacrynate and dihydroethacrynate hardly affect the 2.4 DNP stimulated ATPase activity. Preincubation with succinate or ADP strongly increases the ethacrynate inhibition whereas it decreases dihydroethacrynate inhibition. Ethacrynate and dihydroethacrynate do not affect the efflux of Pi produced by ATP hydrolysis but ethacrynate enforces the inhibitory effect of mersalyl (Mg2+ containing medium). After ten minutes of preincubation with mitochondria, ethacrynate binds 25 nmoles of -SH/mg protein (DTNB titration) and dihydroethacrynate has no effect. These results show an effect of ethacrynate on two types of thiols linked with energy conservation mechanisms and ADP translocation. These thiols could be unmasked or made accessible by conformational modifications of the inner membrane upon energization or addition of ADP.

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.

Dependency of the 2,4-dinitrophenol stimulated ATPase activity on K + and respiration

The 2,4-dinitrophenol (DNP) stimulated ATPase activity has been studied in rat liver mitochondria which have a K + content of 15 mM or less. It has been found that in these mitochondria the ATPase activity becomes strictly dependent on K + and on electron transport. The activity reaches a maximum with 20 mM KCl and is inhibited by rotenone and antimycin. Succinate, in some experiments, enhances the DNP stimulated ATPase activity in a malonate sensitive process.

Biochemical and electrophysiological aspects of neuronal recovery after prolonged ischemia of the cat.

Complete cerebral ischemia of 30min duration was produced in normothermic cats by clamping the innominate and the left subclavian arteries and simultaneously lowering the systemic blood pressure. After ischemia the brain were recirculated with blood for periods of 15 to 2hrs. The suppression and recovery of the neuronal function during and after ischemia were assessed in the sensorimotor cortex by recording the spontaneous EEG and the pyramidal response after stimulation of the cortex; by measuring the concentration of energy metabolites, such as phosphocreatine, adenosine triphosphate, glucose, and lactate, and by measuring the DNP-stimulated ATP-ase activity of isolated mitochondria in the brain. The EEG was suppressed 11±1.4 seconds after the onset of ischemia and the pyramidal response after 240±38 seconds. At the end of ischemia, the enregy rich phosphates were completely exhausted, the glucose content had fallen to 4% of the control value, and lactate increased 23 fold. The DNP-stimulated ATP-ase activity declined 22% of the control value. The ultrastructural alteration of the sensorimotor cortex was minimum at the end of ischemia.Recovery of the pyramidal response started 3 to 6min after restoring the blood flow and was completed within 2hrs; the EEG reappeared after 1 to 2hrs. The functional recovery was related to the restoration of the energy rich phosphates and glucose-levels and to the simultaneous reduction of the accummulated lactate. Under the potimal conditions, CrP and glucose were normalized within 30min and lactate was reduced to 127% of the control value within 2hrs after the end of ischemia. The ATP concentration, and DNP-stimulated ATP-ase activity, however, returned only to 72% and less than 90% respectively, even when spontaneous EEG activity reappeared. The mitochondrial swelling which was observed as one of the structural changes of the sensorimotor cortex taken at this time, seemed to be related to the incomplete recovery of ATP concentration and DNP-stimulated ATP-ase activity.The investigation of the clinical signs of the post-recirculation recovery of the cats was carried out only with conventional nursing of the postischemic animals and no complete recovery of the clinical signs could be observed in these animals.

Inhibition of electron and energy transfer in mitochondria by 19-nor-ethynyltestosterone acetate

19-Nor-ethynyltestosterone acetate (NEA) is an effective inhibitor of mitochondrial respiration in the vicinity of the NADH dehydrogenase flavoprotein. At this site, half-maximal inhibitory values allow the establishment of NEA as a more potent inhibitor than barbiturates and guanidines but less effective than piericidin and rotenone. NEA inhibits both electron and energy transfer in mitochondria and submitochondrial particles, as shown by (a) the higher effectiveness on reversed (energy-dependent) than on forward electron transfer and (b) the partial release of NEA inhibition by uncouplers. Inhibition of energy transfer prevails at the lower (< 20 μ m) NEA concentration and is reversed by serum albumin, while inhibition of electron transfer prevails at the higher (> 30 μ m) NEA concentrations and is apparently irreversible. In contrast to typical inhibitors of energy transfer, NEA releases latent mitochondrial ATPase and does not inhibit the DNP-stimulated ATPase activity. Addition of NEA to mitochondria respiring NAD-linked substrates shifts the redox level of pyridine nucleotide toward the reduced state while the reverse occurs with cytochrome b and ubiquinone. The effect of NEA on absorbancy levels at the flavo-protein spectral region, with both mitochondrial and submitochondrial preparations, can be explained in terms of interaction of NEA with a steroid-sensitive site located on the oxygen side of the mitochondrial NADH flavoprotein, very close or partly superimposed upon the rotenone site. Another flavoprotein, the succinate dehydrogenase, appears to be located between the NEA-sensitive site and the cytochrome chain. As with rotenone, barbiturates and piericidin, NEA inhibition would involve a lipid component of the respiratory chain.

Adenosine triphosphatase activity of heart mitochondria of the turtle, Chrysemys picta

1. 1. ATPase activity has been estimated in heart mitochondria of the fresh-water turtle, Chrysemys picta. 2. 2. This activity is Mg 2+-dependent and stimulated by the presence of DNP. 3. 3. The pH optima of ATPase activities are 8·5 with Mg 2+ ions, and 7·4 in the presence of DNP. 4. 4. Differential responses to inhibitors are evident in the presence of added Mg 2+ ions or DNP, with DNP-stimulated activities being sensitive to a wider spectrum of inhibitors and to the aging process. 5. 5. Bovine serum albumin, but not EDTA, effectively preserves DNP-stimulated ATPase activity during aging. A causal relationship with fatty acids is implied. 6. 6. These data may be used to support either a one or a two enzyme hypothesis, with the latter being favored.

The dependence of oxidative phosphorylation and ATP-ase of liver mitochondria on bound Mg 2+

The concentration of hydrophobic proton donor type uncouplers necessary to to induce maximal ADP dependent ejection of bound mitochondrial Mg 2+ coincides with the minimal concentration of these agents required to uncouple oxidative phosphorylation. DNP stimulated ATP-ase activity of mitochondria and ADP + DNP dependent ejection of bound Mg 2+ exhibit an inverse relationship, i.e. ATP-ase activity depends on bound mitochondrial Mg 2+. It is concluded that bound mitochondrial Mg 2+ is an obligatory component of the mitochondrial energy transducing apparatus.

The nature of the oxidation states of sweet potato mitochondria

Sweet potato mitochondria exhibited respiratory control during the oxidation of malate and succinate with ADP/O ratios approaching the theoretical P/O values. Prior to the addition of ADP the mitochondria showed a considerable rate of substrate oxidation, defined as the basic respiration, which was of the same magnitude as state 4 respiration. Electrons from state 4 and the basic respiration were at least partially mediated by the cytochrome chain, as shown by effects of cyanide, azide and amytal, and by spectrophotometric evidence. The nature of ATPase was studied and the influence of inhibitors of ATPase activity on oxidation helped to establish the relationship between the several states of oxidation and ATPase activity. The ADP/O ratio and ADP-stimulated respiration were slightly decreased by fluoride, while state 4, the basic respiration and ATPase activity were effectively inhibited. Chlorpromazine inhibited DNP-stimulated ATPase activity, respiration uncoupled by DNP and all the states of malate oxidation. However, state 4 and basic respiration were less sensitive than was state 3 of malate oxidation to 0.3 mM chlorpromazine. It was concluded that mitochondrial ATPase played a role in the basic respiration and in state 4 oxidation.

Carbonyl groups associated with mitochondrial ATP synthesis

In an attempt to define the chemical pathway responsible for ATP formation in mitochondria via oxidative phosphorylation, rat liver mitochondria were incubated with chemical compounds that are known to react with carbonyl groups. The effects of the compounds on P i-ATP exchange, ATPase and DNP-stimulated ATPase activity were determined. Hydroxylamine, semicarbazide, and cyanide were found to inhibit P i-ATP exchange activity and DNP-stimulated ATPase activity. Semicarbazide, labeled with carbon 14, was found to bind to mitochondria and the amount bound was dependent on the mitochondrial pretreatment procedure. Our results indicate that there is a functional carbonyl group(s) associated with mitochondrial ATP synthesis and with the reversal of electron transport by ATP.

Inhibition of energy transfer reactions in mitochondria by hydroxylamine

The effect of hydroxylamine on electron and energy transfer reactions has been investigated in rat liver mitochondria with the following results: 1. 1. Succinate oxidation is slightly inhibited by hydroxylamine in the absence of phosphate acceptor (state 4) and in the presence of uncoupler, but is strongly inhibited in the presence of a phosphate acceptor (state 3). Similar effect is observed with other substrates with the extent of inhibition increasing in the order of succinate > ascorbate > NAD-linked substrates. Hydroxylamine strongly inhibits soluble cytochrome oxidase activity but only partially retards terminal electron transfer in mitochondria. 2. 2. Hydroxylamine decreases the rate of energy transfer reactions such as reversed electron transfer, ion translocation, and ATP formation with Ki of 2 mM during succinate oxidation. ATP- 32Pi exchange reaction and DNP-stimulated ATPase activity are not affected by hydroxylamine. 3. 3. Hydroxylamine does not inhibit the oxidation of fatty acids in the presence of carnitine, ATP, and uncoupler, suggesting that the reagent is not reacting with acyl compounds in mitochondria. It is concluded that hydroxylamine interacts at an early, as yet unidentified, energy transfer step in mitochondria.

Inhibition of oxidative phosphorylation and related reactions in insect mitochondria

1.1. The effects of the uncoupling agent 2,4-dinitrophenol and the insecticide DDT on respiration, phosphorylation, and the ATPase and ATP-Pi exchange activities of housefly mitochondria were investigated.2.2. DDT at 0.1 mM abolishes phosphorylation, the ATP-Pi exchange, and the DNP-stimulated ATPase activity in these mitochondria. DDT does not, however, affect the Mg2+-stimulated ATPase activity; in this respect it appears to be unique among known uncouplers or inhibitors of mitochondrial ATPase.3.3. DDT also inhibits that portion of the total respiration which is “tightly-coupled” to oxidative phosphorylation; “uncoupled” respiration is not depressed by DDT.4.4. Anomalously, DNP frequently strongly inhibits respiration in the presence of ADP and Pi and fails to stimulate pyruvate oxidation in the absence of ADP and Pi.

Preservation of washed bovine spermatozoa in a synthetic medium at room temperature

1. 1. Development is described of a completely synthetic medium that permitted storage at room temperature of washed bovine spermatozoa for one week with 10 per cent loss of initial motility. 2. 2. The respiratory ability and the ATP and ADP content of the stored cells declined rapidly with time. The relationship of these measurements to the state of “health” of the cells is not clear. 3. 3. Measurement of DNP-stimulated ATP-ase activity of stored cells indicated that the ability to couple oxidation and phosphorylation may not be impaired.

The relation of the site of action of amytal to oxidative phosphorylation

19-Nor-ethynyltestosterone acetate (NEA) is an effective inhibitor of mitochondrial respiration in the vicinity of the NADH dehydrogenase flavoprotein. At this site, half-maximal inhibitory values allow the establishment of NEA as a more potent inhibitor than barbiturates and guanidines but less effective than piericidin and rotenone. NEA inhibits both electron and energy transfer in mitochondria and submitochondrial particles, as shown by (a) the higher effectiveness on reversed (energy-dependent) than on forward electron transfer and (b) the partial release of NEA inhibition by uncouplers. Inhibition of energy transfer prevails at the lower (< 20 μm) NEA concentration and is reversed by serum albumin, while inhibition of electron transfer prevails at the higher (> 30 μm) NEA concentrations and is apparently irreversible. In contrast to typical inhibitors of energy transfer, NEA releases latent mitochondrial ATPase and does not inhibit the DNP-stimulated ATPase activity. Addition of NEA to mitochondria respiring NAD-linked substrates shifts the redox level of pyridine nucleotide toward the reduced state while the reverse occurs with cytochrome b and ubiquinone. The effect of NEA on absorbancy levels at the flavo-protein spectral region, with both mitochondrial and submitochondrial preparations, can be explained in terms of interaction of NEA with a steroid-sensitive site located on the oxygen side of the mitochondrial NADH flavoprotein, very close or partly superimposed upon the rotenone site. Another flavoprotein, the succinate dehydrogenase, appears to be located between the NEA-sensitive site and the cytochrome chain. As with rotenone, barbiturates and piericidin, NEA inhibition would involve a lipid component of the respiratory chain.