ATPase Activity Of Submitochondrial Particles Research Articles

Stoichiometry of subunit e in rat liver mitochondrial H +-ATP synthase and membrane topology of its putative Ca 2+-dependent regulatory region

Previous studies have revealed that residues 34–65 of subunit e of mitochondrial H +-ATP synthase are homologous with the Ca 2+-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca 2+-dependent regulation of H +-ATP synthase activity. In this study, we determined the content of subunit e in H +-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca 2+-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34–65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H +-ATP synthase revealed that 1 mol of H +-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F 0 is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H +-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca 2+-dependent regulatory region of subunit e is exposed on the surface of the C-side of F 0 and that subunit e is involved in the regulation of mitochondrial H +-ATP synthase activity probably via its putative Ca 2+-dependent regulatory region.

Dibutyltin‐3‐hydroxyflavone titrates a dissociable component (cofactor) of mitochondrial ATP synthase: An energy‐transfer component linked to the ubiquinone pool

AbstractDibutyltin‐3‐hydroxyflavone, Bu2Sn(of), is a new fluorescence probe inhibitor of F1F0‐ATPase and oxidative phosphorylation which inhibits by titration of an unidentified component of F0. Its site of action is closely related to that of the trialkyltins and of venturicidin. This F0 component is part of a pool of this component which is present in the heart mitochondrial inner membrane at levels of 5–7 nmol (mg protein)−1 [18 ± 3 Bu2Sn(of) sites per mol F1F0‐ATPase]. However, ATPase activity in submitochondrial particles is near maximally inhibited by titration of approx. three Bu2Sn(of) sites per mol F1F0‐ATPase.Over 60% (60–80%) of the Bu2Sn(of) interaction sites can be lost during the purification of F1F0‐ATPase from submitochondrial particles. The number of Bu2Sn(of) interaction sites in various F1F0‐ATPase preparations is variable. The high numbers of Bu2Sn(of) sites per mol F1F0‐ATPase for heart mitochondria (18–21) and submitochondrial particles (15–19.5) decline in ATP synthase (11–15) to the low values obtained in Complex V (7–10.5) and the minimal values observed in highly purified F1F0−ATPase (3.5–5.6), thus indicating a variable dissociable component or cofactor of ATP synthase.The Bu2Sn(of) interaction site, a component of ATP synthase, is responsive to the redox status of the respiratory chain and the interaction with Bu2Sn(of) is with the reduced form of this component. Fluorescence titration studies show that this component is in redox equilibrium with the ubiquinone pool of the respiratory chain. It is proposed that this redox component serves as an inhibitor titratable cofactor pool which cycles through an F0 interaction site (or sites) via a system which serves as an energy‐transfer link between the respiratory chain and ATP synthase.

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.

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 1 mM MgCl2. 8. The observed effects of gossypol could be explained by the collapse of the mitochondrial membrane potential.

Inhibition by suramin of mitochondrial ATP synthesis

Suramin, a drug intensively used in the chemotherapy of African trypanosomiasis and onchocerciasis, is currently being tested in clinical trials for AIDS treatment. Its effects on mitochondrial energy metabolism in mammals were studied. At low concentrations it inhibited ATP synthesis and ATPase activity in submitochondrial particles, as well as ADP-stimulated oxygen consumption and the uncoupler-stimulated ATPase activity in intact rat liver mitochondria. At higher concentrations it also inhibited uncoupled electron transport in both submitochondrial particles and intact mitochondria. From comparison of the kinetic patterns of those inhibitions, evidence suggesting that the adenine nucleotide translocase may be another target for the action of suramin was obtained. The relevance of these findings to the understanding of the biochemical basis of suramin toxicity is discussed.

Inhibition of mitochondrial energy-linked reactions by 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (dimboa), a hydroxamic acid from gramineae

DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) is the main hydroxamic acid isolated from maize extracts. It inhibited reversibly ATP synthesis, P I-ATP exchange reaction and ATPase activity in submitochondrial particles from bovine heart. Half-maximal effects were obtained with 4, 2, and 6 mM DIMBOA respectively. At higher concentrations it also inhibited mitochondrial electron transport ( I 50 = 11 mM). Irreversible inactivation of mitochondrial electron transport, P i-ATP exchange reaction and 8-anilino-1-naphthalene sulfonate energy-dependent fluorescence enhancement was also observed. These effects of DIMBOA on energy-linked mitochondrial reactions may explain the inhibitory action of DIMBOA on several aerobic organisms.

Studies on polypeptide composition, hydrolytic activity and proton conduction of mitochondrial FoF1 H+ ATPase in regenerating rat liver.

A study of the FoF1 ATPase complex of mitochondria isolated from regenerating rat liver following partial (70%) hepatectomy is presented. As we have previously reported, ATPase activity in submitochondrial particles prepared from regenerating rat liver 24 h following partial hepatectomy was depressed by 75% with respect to controls (submitochondrial particles from sham-operated animals). Polyacrylamide gel electrophoresis and immunodecoration using an antibody raised against isolated bovine heart F1 sector of the FoF1 ATPase indicated a substantial decrease in F1 content in the mitochondrial membrane from regenerating rat liver. Proton conduction by the FoF1 ATPase complex was studied by following the anaerobic relaxation of the transmembrane proton gradient (delta mu H+) generated by succinate-driven respiration. In control rat-liver submitochondrial particles containing the FoF1 moiety of the ATPase complex, anaerobic relaxation of delta mu H+ showed biphasic kinetics, whilst the same process in particles derived from regenerating rat liver exhibited monophasic kinetics and was significantly more rapid. Oligomycin and N,N-dicyclohexyl carbodiimide [(cHxN)2C] inhibited proton conductance by the F1-Fo ATPase complex in submitochondrial particles from both control and regenerating rat liver. Binding of [14C](cHxN)2C and immunodecoration using an antibody raised against bovine heart oligomycin-sensitivity-conferring protein (OSCP) indicated no difference in the content of either the (cHxN)2C binding protein or OSCP between control and regenerating rat-liver mitochondrial membranes. The results reported show that the structural and functional integrity of the Fo-F1 ATPase of rat liver is severely perturbed during regeneration.

Inhibition of the mitochondrial Mg 2+-ATPase by propranolol

The in vitro effects of propranolol, a commonly used β-adrenergic blocker, on the membrane structure and function of rat heart mitochondria were investigated. It was found that the respiratory control and oxidative phosphorylation of the isolated mitochondria decreased concomitantly when the drug was added to the assay medium. At the concentration higher than 1.0 × 10 −4 M, propranolol significantly inhibited the State 3 respiration but had little effect on the State 4 respiration of the mitochondria. On the other hand, the drug exhibited noncompetitive inhibitions toward the Mg 2+-ATPase activity of submitochondrial particles and purified enzyme preparations at the concentrations ranging from 3.0 × 10 −4 to 1.5 × 10 −3 M. The inhibitory constants of propranolol toward the enzyme activity in submitochondrial particles and in the purified preparation were estimated to be 6.7 × 10 −4 and 1.4 × 10 −3 M, respectively. However, the drug did not show significant effect on the activity of any of the enzyme complexes of the mitochondrial respiratory chain. It is thus concluded that propranolol impairs the mitochondrial respiration and oxidative phosphorylation mainly through its inhibition of the Mg 2+-ATPase activity of the mitochondria. This effect of propranolol may explain, at least partly, its depression effects on the cardiac functions of the animal.

Effect of the protonmotive force on ATP-linked processes and mobilization of the bound natural ATPase inhibitor in beef heart submitochondrial particles

In an attempt to determine whether the natural ATPase inhibitor (IF1) plays a role in oxidative phosphorylation, the time course of ATP synthesis and ATP hydrolysis in inside-out submitochondrial particles from beef heart mitochondria either possessing IF1 (Mg-ATP particles) or devoid of IF1 (AS particles) was investigated and compared to movements of IF1, as assessed by an isotopic assay. The responses of the above reactions to preincubation of the particles in aerobiosis with NADH or succinate were as follows: (1) The few seconds lag that preceded the steady-rate phase of ATP synthesis was shortened and even abolished both in Mg-ATP particles and AS particles. The rate of ATP synthesis in the steady state was independent of the length of the lag. (2) ATPase was slowly activated, maximal activation being obtained after a 50-min preincubation; there was no direct link between the development of the protonmotive force (maximal within 1 sec) and ATPase activation. (3) Bound IF1 was slowly released; the release of bound IF1 as a function of the preincubation period was parallel to the enhancement of ATPase activity; the maximal amount of IF1 released was a small fraction of the total IF, bound to the particles (less than 20%). (4) The double reciprocal plots of the rates of ATP and ITP hydrolysis vs. substrate concentrations that were curvilinear in the absence of preincubation with a respiratory substrate became linear after aerobic preincubation with the substrate. The data conclusively show that only ATPase activity in submitochondrial particles is correlated with the release of IF1, and that the total extent of IF1 release induced by respiration is limited. On the other hand, the kinetics of ATPase in control and activated particles are consistent with the existence of two conformations of the membrane-bound F1-ATPase, directed to ATP synthesis or ATP hydrolysis and distinguishable by their affinity for IF1.

Inhibition of oxidative phosphorylation by an oligomer of prostaglandin B 1, PGB x

PGB x is a synthetic, oligomeric derivative of prostaglandin B 1 that has been shown to protect rat liver mitochondria from the deleterious effects of aging. In fresh mitochondria, PGB x inhibits reactions involving the F 1F 0-ATPase. It prevents the stimulation of respiration by ADP and inhibits ATP-driven Ca 2+ transport. It has no effect, however, on Ca 2+-stimulated respiration and associated proton movements, or on respiration-driven Ca 2+ transport, indicating that PGB x is not an inhibitor of the electron transport chain. The ATPase activity of submitochondrial particles is inhibited by PGB x with mixed type kinetics in which both K m and V are affected. PGB x has no effect on the ATPase activity of soluble F 1, but induces respiratory control in F 1-deficient submitochondrial particles, indicating a mode of action similar to oligomycin and DCCD. The binding of DCCD to its proteolipid receptor is inhibited by PGB x, suggesting that this is the binding site for PGB x. It is concluded that PGB x inhibits reactions involving the F 1F 0-ATPase by binding at or near the DCCD-binding protein and blocking proton conduction through the F 0 moiety of the complex.

Oxygen uptake, ATPase activity, and superoxide dismutase activity in isolated rat liver mitochondria are not influenced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) influences neither the State 3 nor the State 4 respiration in rat liver mitochondria. The respiratory control and ADP/O ratio were also unaffected by TPA. The oligomycin-sensitive ATPase activity in submitochondrial particles remained unaltered upon TPA addition, whereas the NADH oxidase activity was slightly inhibited at a very high concentration of TPA (15% decrease at 17 microM TPA). The activity of the superoxide dismutase located to the mitochondria was insensitive to the tumor promoter, and no change in the rate of H2O2 production was found on TPA treatment in vitro. Thus, the mitochondrion is not a likely candidate for the site of action of the tumor promoter.

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Partial characterization and changes in activity during exponential growth

1. 1. The mitochondrial adenosine triphosphatase (ATPase) of Acanthamoeba castellanii is Mg 2+-requiring (optimum cation: ATP ratio of 1.5) and has two pH optima of activity (at pH 6.6 and 8.1). 2. 2. ATPase activity of submitochondrial particles is effectively inhibited by twelve different inhibitors of energy conservation suggesting similarities in inhibitor-binding sites to other previously characterized complexes. 3. 3. Gel filtration by passage through Sephadex G-50 increases ATPase activity of submitochondrial particles between 1.5 and 3.5 fold indicating the presence of a low molecular weight inhibitor protein. 4. 4. After removal of the inhibitor protein, sensitivity to inhibitors of energy conservation decreases by between 1.5 and 14 fold. Crude F 1- inhibitor preparations from A. castellanii, Schizosaccharomyces pombe, Tetrahymena pyriformis and bovine heart also inhibit ATPase activity. 5. 5. Large variations in ATPase activity, F 1- inhibitor protein activity, and amounts of immunologically-determined ATPase protein were observed during exponential growth, and the correlation between changes in these measurements is discussed. 6. 6. The results are also discussed highlighting the similarities between the mitochondrial ATPase of A. castellanii and other mitochondrial ATPases.

MgATP-induced inhibition of the adenosine triphosphatase activity of submitochondrial particles.

1. The ATP-hydrolytic activity of ox heart submitochondrial particles can be increased from 2-3 mumol/min per mg of protein to 10-12 mumol/min per mg of protein by incubation in media containing 50 mM-Na2B4O7. This process appears to be due to the partial release of inhibitor protein from the particles. 2. The ATPase activity of submitochondrial particles can be inhibited by incubation with the substrate, MgATP. This inhibition is not due to the accumulation of the hydrolysis products, MgADP and Pi, but could involve the process of ATP hydrolysis. 3. The mechanism of MgATP-induced inhibition of ATPase activity is proposed to involve a conformational change in one of the intermediate enzyme species of the ATP-hydrolytic sequence. 4. MgATP inhibits the ATPase activity of control submitochondrial particles at a higher rate and to a greater extent than it does that of inhibitor-protein-depleted submitochondrial particles, suggesting that the conformational change involves the endogenous inhibitor protein.

Selective disaggregation of the H+-translocating ATPase. Isolation of two discrete complexes of the rutamycin-insensitive ATPase differing in mitochondrial membrane-binding properties.

The H+-translocating ATPase from rat liver mitochondria can be disaggregated selectively to yield two distinct, stable complexes of the rutamycin-insensitive ATPase. The two ATPase complexes can be purified to homogeneity by zone sedimentation in a glycerol gradient. Based on their electrophoretic mobility in 5% polyacrylamide gels, the aggregates have been designated as type I (Rf = 0.49) ATPase and type II (Rf = 0.56) ATPase. These two complexes of the ATPase differ in ATP hydrolytic activity, in stability, in mobility on 5% polyacrylamide gel electrophoresis, in subunit composition, and in ability to reassociate with submitochondrial particles which are highly depleted in ATPase activity. The type II ATPase is similar to the F1-ATPase, but the type I ATPase contains a 26.5-kilodalton subunit not present in the type II enzyme. This 26.5-kilodalton subunit is equimolar with the gamma subunit of the ATPase (based on Coomassie blue dye binding); its presence seems to be correlated to the altered properties of the type I ATPase. Type I ATPase reconstitutes rutamycin-sensitive ATPase activity in submitochondrial particles treated with trypsin, urea, ammonia, and 1.5% silicotungstic acid. The type II ATPase does not reconstitute rutamycin-sensitive ATPase activity in these ATPase-depleted submitochondrial particles unless it is supplemented with the 26.5-kilodalton subunit isolated from the type I ATPase. The 26.5-kilodalton protein has thus been functionally identified as important for the binding of the ATPase to the membrane by providing a direct link to the membrane or by binding to the ATPase putting it in an appropriate conformation for binding.

Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles.

The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase.

Mixed anhydrides of nucleotides and mesitylenecarboxylic acid as new specific inhibitors of mitochondrial adenosien triphosphatase

Mixed anhydrides of nucleoside triphosphates and mesitylenecarboxylic acid inhibit soluble mitochondrial ATPase (adenosine triphosphatase), but do not inhibit ATPase of submitochondrial particles. Inhibition of soluble mitochondrial ATPase by the mixed anhydride of epsilon-ATP and mesitylenecarboxylic acid is followed by the covalent binding of one nucleotide residue to a molecule of the protein. It is suggested that this covalent binding occurs in the catalytic site of the mitochondrial ATPase. The mixed anhydride of ADP and mesitylenecarboxylic acid inhibits the ATPase activity of submitochondrial particles and has no effect on the activity of soluble mitochondrial ATPase. After separation of the submitochondrial particles from the mixed anhydride of ADP and mesitylenecarboxylic acid, their ATPase activity is restored to its original value (half-time of reactivation 3--4 min). Incubation of submitochondrial particles or soluble mitochondrial ATPase with the mixed anhydride of ADP and mesitylenecarboxylic acid results in AMP formation.

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 F 1 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 F 1 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.

On the mechanism of action of alkylguanidines in oxidative phosphorylation: Their action on soluble F 1

Octylguanidine inhibits the adenosine triphosphatase (ATPase) activity of bovine heart submitochondrial particles and soluble F 1. The characteristics of the inhibition as a function of octylguanidine and Mg 2+ concentrations and pH are very similar in submitochondrial particles and soluble F 1. Only those guanidines that possess an alkyl chain of more than six carbons inhibit the ATPase activity of submitochondrial particles and F 1. The inhibiting action of octylguanidine on F 1 is fully reversible. Octylguanidine prevents the cold-induced inactivation of F 1 at concentrations similar to those that inhibit ATPase activity. Guanidines that inhibit ATPase activity also prevent the cold-induced inactivations of F 1.

A new inhibitor of coupled oxidative phosphorylation, 5-hydroxynaphthalenedicarboxylic anhydride, a derivative of a carcinogenic polynuclear hydrocarbon.

5-Hydroxy-1,2-naphthalenedicarboxylic anhydride is closely related to its precursor dibasic acid which is a metabolite of the carcinogenic polynuclear hydrocarbon dibenz[a,h]anthracene. The anhydride inhibited respiration of coupled mitochondria. This inhibition was relieved by 2,4-dinitrophenol. Several mitochondrial volume change processes energized by ATP were also inhibited by the anhydride. Both the mitochondrial ATPase activity induced by 2,4-dinitrophenol and the ATPase activity of submitochondrial particles induced by magnesium ion were inhibited by the anhydride. The spectrum of inhibitory activity was not associated with acetic anhydride, succinic anhydride, or phthalic anhydride. The data indicate that 5-hydroxy-1,2-naphthalenedicarboxylic anhydride inhibits the machinery of oxidative phosphorylation in a manner similar to rutamycin. 5-Hydroxy-1,2-naphthalenedicarboxylic anhydride is the first molecule derived from a carcinogen with such inhibitory properties.

The Mitochondrial ATPase

1. Evidence is presented which indicates that inactivation of the mitochondrial ATPase from bovine heart by the reagent 4-chloro-7-nitrobenzofurazan results from modification of one tyrosine residue per enzyme molecule. Activity can be restored by a variety of sulphydryl reagents. 2. In sodium dodecyl sulphate, the nitrogenzofurazan group on tyrosine is transfered to newly exposed sulphydryl groups on the enzyme. 3. The rate of transfer of the nitrobenzofurazan moiety from theenzyme to sulphydryl compounds is compared with that for transfer from the model compound N-acetyl-tyrosine-0(7-nitrobenzo-furazan) ethyl ester, the synthesis and properties of which are also described. 4. The ligands ATP and ADP exert a protective effect on the rate of reaction between the mitochondrial ATPase and 4-chloro-7-nitrobenzofurazan. The variation in rate of this reaction with change in pH has also been examined and a pKa of 9.5 estimated for the tyrosine residue. 5. The modification does not prevent substrate binding as judged by changes in the fluorescence of aurovertin, an antibiotic with specific affinity for mitochondiral ATPases. 6. When the ATPase activity of submitochondrial particles is inhibited by 4-chloro-7-nitrobenzo-furazan, there is a parallel decrease in the extent of the energy-linked fluorescence enhancement of 1-anilino-naphthalene-8-sulphonate induced by ATP hydrolysis. Both ATPase activity and the fluorescence enhancement are restored by sluphydryl reagents.