F1F0 ATP Synthase Activity Research Articles

Epileptiform Discharges Reduce Neuronal ATP Production by Inhibiting F0F1-ATP Synthase Activity via A Zinc-α2-Glycoprotein-Dependent Mechanism.

Neuronal energy metabolism dysfunction, especially adenosine triphosphate (ATP) supply decrease, is observed in epilepsy and associated with epileptogenesis and prognosis. Zinc-α2-glycoprotein (ZAG) is known as an important modulator of energy metabolism and involved in neuronal glucose metabolism, fatty acid metabolism, and ketogenesis impairment in seizures, but its effect on neuronal ATP synthesis in seizures and the specific mechanism are unclear. In this study, we verified the localization of ZAG in primary cultured neuronal mitochondria by using double-labeling immunofluorescence, immune electron microscopy, and western blot. ZAG level in neuronal mitochondria was modulated by lentiviruses and detected by western blot. The F0F1-ATP synthase activity, ATP level, and acetyl-CoA level were measured. The binding between ZAG and F0F1-ATP synthase was determined by coimmunoprecipitation. We found that both ZAG and F0F1-ATP synthase existed in neuronal mitochondria, and there was mutual binding between them. Epileptiform discharge-induced decrease of mitochondrial ZAG level was reversed by ZAG overexpression. Epileptiform discharge or ZAG knockdown decreased F0F1-ATP synthase activity and ATP level in neurons, which were reversed by ZAG overexpression, while overexpression of ZAG along only increased F0F1-ATP synthase activity but not increased ATP level. Meanwhile, neither epileptiform discharges nor changes of ZAG level can alter the acetyl-CoA level. Moreover, epileptiform discharge did not alter F0F1-ATP synthase level. In conclusion, epileptiform discharge-induced ZAG decrease in neuronal mitochondria is correlated to F0F1-ATP synthase activity inhibition, which may possibly lead to ATP supply impairments. ZAG may be a potential therapeutic target for treating neuronal energy metabolism dysfunction in seizures with further researches.

Dietary Ginger Phenolics Potent Inhibition of E. coli ATP Synthase

The broad long-term goal of our lab is to find and characterize potent selective inhibitors of ATP synthase. ATP synthase is the main source of cellular energy in all organisms. Selective inhibition of bacterial ATP synthase may be utilized to fight the drug-resistant microbial infections. Many phytochemicals, including phenolics, are known to bind at the phytochemical binding site of ATP synthase. In the current study, we are interested in the effect of dietary ginger phenolics (DGPs) on bacterial ATP synthase. DGPs exhibit antimicrobial and anticancer properties similar to many other categories of phytochemicals. Previously our lab has documented that a variety of dietary phytochemicals have selective inhibitory effects on ATP synthase. This leads to the experimental question of this project: What is the effect of DGP on E. coli cell growth and ATP synthase activity? This question will be further addressed in the following specific aims. Specific Aim 1: To determine the effects of DGP on E. coli membrane bound F1F0 ATP synthase activity. Wild type and mutant E. coli enzymes will be isolated and examined for activity of the enzyme. Assays of E. coli strains will be conducted in the presence of DGPs. DGP-induced inhibitory profiles will yield a dose response curve measured at OD595. ATP synthase activity should decrease as the concentration is increased for each mutant strain, as well as in the presence of the ginger phenolics. Specific Aim 2: To determine the effects of DGP on growth in wild type and mutant E. coli strains. Wild type, null, and mutant E. coli strains will be grown in presence and absence of DGP. Growth will be measured on fermentable limiting glucose and non-fermentable succinate media. Abrogation of growth in presence of DGP will be compared to isolated E. coli ATP synthase growth on different media. The novel aspect of this research is the selective inhibition of ATP synthase in bacteria. The selective inhibition could ultimately help combat the issue of increasing antibiotic resistant microbial infections.

A mathematical model for persistent post-CSD vasoconstriction.

Cortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity.

Inorganic polyphosphate is produced and hydrolyzed in F0F1-ATP synthase of mammalian mitochondria.

Inorganic polyphosphate (polyP) is a polymer present in all living organisms. Although polyP is found to be involved in a variety of functions in cells of higher organisms, the enzyme responsible for polyP production and consumption has not yet been identified. Here, we studied the effect of polyP on mitochondrial respiration, oxidative phosphorylation and activity of F0F1-ATPsynthase. We have found that polyP activates mitochondrial respiration which does not coupled with ATP production (V2) but inhibits ADP-dependent respiration (V3). Moreover, PolyP can stimulate F0F1-ATPase activity in the presence of ATP and, importantly, can be hydrolyzed in this enzyme instead of ATP. Furthermore, PolyP can be produced in mitochondria in the presence of substrates for respiration and phosphate by the F0F1-ATPsynthase. Thus, polyP is an energy molecule in mammalian cells which can be produced and hydrolyzed in the mitochondrial F0F1-ATPsynthase.

Abstract 801: Dual inhibition of distinct metabolic features targets osteosarcoma stem cells

Abstract [Objective] Mitochondria are the places for the energy production of the cells, while reactive oxygen species (ROS) are also produced alongside. In recent years, it has been reported that cancer stem cells metabolize predominantly through oxidative phosphorylation (OXPHOS) rather than glycolysis in certain cancer cells. Targeting OXPHOS achieved by suppression of ATP synthesis through mitochondrial ATP synthase could be a potential therapeutic option against cancer stem cells. In the current study, we have identified the mitochondria metabolism as the potential therapeutic target in osteosarcoma (OS) stem cells, presenting the synergistic effects of combination of OXPHOS inhibition by pterostilebene (PTE) with c-Myc inhibitor, which target both OXPHOS-dominant cancer stem cells and glycolysis-dominant non-cancer stem cells as a ‘two hit’ or ‘dual inhibition’ of metabolic pathways, OXPHOS and glycolysis. [Materials & Methods] Using human OS cell lines of SaOS2, U2OS and MG63 cells, cell survival and the ability of sphere formation was assessed with or without PTE, and the expression of stem cell markers mRNA such as Oct3, NS, CD44 was examined by RT-PCR. Next, the activity of mitochondrial ATP synthase, mitochondrial respiration capacity of oxygen consumption rate, and the amount of ATP as well as ROS production were measured under the treatment of PTE. Furthermore, we examined the synergistic effect of PTE with cMyc transcription inhibitors of JQ1 or Honokiol (HNK). [Results] PTE treatment on human OS cell lines reduced the viabilities of all cell lines in dose-dependent manner and expression of stem cell marker and the ability of sphere formation were also decrease in terms of sphere number and size. PTE reduced the activity of F0F1-ATP synthase; Complex V predominantly, and the mitochondrial oxygen consumption rates and synthetic amount of ATP were also decreased in spheroid condition. These results suggest that PTE possibly targets stem cell population which preferably relies on OXPHOS, suppressing ATP synthesis via F0F1-ATP synthase inhibition as well as increased ROS production in OS cells and changes metabolic flax to glycolysis dependent feature. The dual inhibition of OXPHOS by PTE and c-Myc inhibition by HNK or JQ1 showed the synergistically inhibition of OS cell growth in a dose-dependent manner. [Discussion] Prognosis of the patients with osteosarcoma has reached to plateau without any breakthroughs over the last quarter century, and nearly 30% of patients still have to face very severe poor prognosis, especially with metastatic disease. Current study suggests that modulation of metabolic flux by c-Myc and OXPHOS inhibitors showed a greater synergistic effect with ‘two metabolic hit’ or ‘dual metabolic inhibition’ of distinct metabolic features and it could be a novel therapeutic strategy against osteosarcoma, possibly targeting both stem-like cell population and general tumor cell population. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Yasuhito Tanaka, Hiroki Kuniyasu. Dual inhibition of distinct metabolic features targets osteosarcoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 801.

Gboxin is an oxidative phosphorylation inhibitor that targets glioblastoma

Cancer specific inhibitors reflective of unique metabolic needs, are rare. We describe a novel small molecule, Gboxin, that specifically inhibits primary mouse and human glioblastoma (GBM) cell growth but not mouse embryo fibroblasts or neonatal astrocytes. Gboxin rapidly and irreversibly compromises GBM oxygen consumption. Reliant on its positive charge, Gboxin associates with mitochondrial oxidative phosphorylation complexes in a proton gradient dependent manner and inhibits F0F1 ATP synthase activity. Gboxin resistant cells require a functional mitochondrial permeability transition pore that regulates pH impeding matrix accumulation. Administration of a pharmacologically stable Gboxin analog inhibits GBM allografts and patient derived xenografts. Gboxin toxicity extends to established human cancer cell lines of diverse organ origin and exposes the elevated proton gradient pH in cancer cell mitochondria as a new mode of action for antitumor reagent development.

The effect of culture conditions on the accumulation and activity of F0F1 ATP synthase in thermophilic bacteria BacillusPS3

AbstractBACKGROUNDThe proton‐translocating protein F0F1 ATP synthase is a transmembrane protein that catalyzes ATP synthesis and is important in artificial photosynthesis and other life science studies. The objective of this study was to understand the accumulation and ATPase activity of F0F1 ATP synthase in the thermophilic bacteria Bacillus PS3 affected by culture conditions, including the type of nitrogen source and the concentrations of polypeptone and sodium chloride in the culture medium, as well as medium pH and aeration rate.RESULTSThe highest yield and ATPase activity of purified enzymes were obtained when adding polypeptone as the nitrogen source at higher concentrations. Medium pH and aeration rate showed significant effects on the accumulation and activity of F0F1 ATP synthase. Contrarily, sodium chloride concentration had little effect on the growth of PS3 or F0F1 ATP synthase accumulation. The highest yield and specific activity of purified F0F1 ATP synthase achieved were 1.631 g L−1 and 7.99 µmol min−1 mg−1, under the culture conditions of pH 7, aeration of 3 L min−1 and 0.3% NaCl and 1.2% (w/w) polypeptone concentrations. The F0F1 ATP synthase purified was characterized by SDS‐PAGE and contained all expected subunits.CONCLUSIONAll of the studied culture conditions except for NaCl concentration significantly affected Bacillus PS3 cell growth and ATP synthase accumulation and reactivity. Polypeptone at higher concentrations in the culture medium along with neutral pH and greater aeration rates were desirable. Cell growth and ATPase activity of F0F1 ATP synthase were highly positively associated. © 2018 Society of Chemical Industry

Negative supercoiling of DNA by gyrase is inhibited in Salmonella enterica serovar Typhimurium during adaptation to acid stress.

DNA in intracellular Salmonella enterica serovar Typhimurium relaxes during growth in the acidified (pH 4-5) macrophage vacuole and DNA relaxation correlates with the upregulation of Salmonella genes involved in adaptation to the macrophage environment. Bacterial ATP levels did not increase during adaptation to acid pH unless the bacterium was deficient in MgtC, a cytoplasmic-membrane-located inhibitor of proton-driven F1 F0 ATP synthase activity. Inhibiting ATP binding by DNA gyrase and topo IV with novobiocin enhanced the effect of low pH on DNA relaxation. Bacteria expressing novobiocin-resistant (NovR ) derivatives of gyrase or topo IV also exhibited DNA relaxation at acid pH, although further relaxation with novobiocin was not seen in the strain with NovR gyrase. Thus, inhibition of the negative supercoiling activity of gyrase was the primary cause of enhanced DNA relaxation in drug-treated bacteria. The Salmonella cytosol reaches pH 5-6 in response to an external pH of 4-5: the ATP-dependent DNA supercoiling activity of purified gyrase was progressively inhibited by lowering the pH in this range, as was the ATP-dependent DNA relaxation activity of topo IV. We propose that DNA relaxation in Salmonella within macrophage is due to acid-mediated impairment of the negative supercoiling activity of gyrase.

Hypoxia and classical activation limits Mycobacterium tuberculosis survival by Akt-dependent glycolytic shift in macrophages.

Cellular reactive oxygen species (ROS) is a major antibacterial defense mechanism used by macrophages upon activation. Exposure of Mycobacterium tuberculosis (Mtb)-infected macrophages to hypoxia is known to compromise the survival of the pathogen. Here we report that the hypoxia-induced control of intracellular Mtb load in RAW 264.7 macrophages was mediated by regulating the cellular ROS levels. We show that similar to classical activation, hypoxia incubation of macrophages resulted in decreased mitochondrial outer membrane potential (MOMP) and a concomitant increase in the cellular ROS levels. Mitochondrial depolarization and consequently higher ROS could be blocked by knocking down Akt using siRNAs, which acted by inhibiting the switch to glycolytic mode of metabolism, an essential adaptive response upon classical activation or hypoxic incubation of macrophages. Moreover, in the classically activated macrophages or in the macrophages under hypoxia incubation, supplementation with additional glucose had similar effects as Akt knockdown. Interestingly, in both the cases, the reversal of phenotype was linked with the ability of the mitochondrial F0–F1 ATP synthase activity to maintain the MOMP in the absence of oxidative phosphorylation. Both Akt knockdown and glucose supplementation were also able to rescue Mtb survival in these macrophages upon classical activation or hypoxia incubation. These results provide a framework for better understanding of how the interplay between oxygen supply, which is limiting in the human tubercular granulomas, and nutrient availability could together direct the outcome of infections in vivo.

Mitochondrial functions of RECQL4 are required for the prevention of aerobic glycolysis-dependent cell invasion

Germline mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome, which is characterized by a predisposition to cancer. RECQL4 localizes to the mitochondria, where it acts as an accessory factor during mitochondrial DNA replication. To understand the specific mitochondrial functions of RECQL4, we created isogenic cell lines, in which the mitochondrial localization of the helicase was either retained or abolished. The mitochondrial integrity was affected due to the absence of RECQL4 in mitochondria, leading to a decrease in F1F0-ATP synthase activity. In cells where RECQL4 does not localize to mitochondria, the membrane potential was decreased, whereas ROS levels increased due to the presence of high levels of catalytically inactive SOD2. Inactive SOD2 accumulated owing to diminished SIRT3 activity. Lack of the mitochondrial functions of RECQL4 led to aerobic glycolysis that, in turn, led to an increased invasive capability within these cells. Together, this study demonstrates for the first time that, owing to its mitochondrial functions, the accessory mitochondrial replication helicase RECQL4 prevents the invasive step in the neoplastic transformation process.

Altered Lipid Synthesis by Lack of Yeast Pah1 Phosphatidate Phosphatase Reduces Chronological Life Span

In Saccharomyces cerevisiae, Pah1 phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid triacylglycerol. This evolutionarily conserved enzyme also plays a negative regulatory role in controlling de novo membrane phospholipid synthesis through its consumption of phosphatidate. We found that the pah1Δ mutant was defective in the utilization of non-fermentable carbon sources but not in oxidative phosphorylation; the mutant did not exhibit major changes in oxygen consumption rate, mitochondrial membrane potential, F1F0-ATP synthase activity, or gross mitochondrial morphology. The pah1Δ mutant contained an almost normal complement of major mitochondrial phospholipids with some alterations in molecular species. Although oxidative phosphorylation was not compromised in the pah1Δ mutant, the cellular levels of ATP in quiescent cells were reduced by 2-fold, inversely correlating with a 4-fold increase in membrane phospholipids. In addition, the quiescent pah1Δ mutant cells had 3-fold higher levels of mitochondrial superoxide and cellular lipid hydroperoxides, had reduced activities of superoxide dismutase 2 and catalase, and were hypersensitive to hydrogen peroxide. Consequently, the pah1Δ mutant had a shortened chronological life span. In addition, the loss of Tsa1 thioredoxin peroxidase caused a synthetic growth defect with the pah1Δ mutation. The shortened chronological life span of the pah1Δ mutant along with its growth defect on non-fermentable carbon sources and hypersensitivity to hydrogen peroxide was suppressed by the loss of Dgk1 diacylglycerol kinase, indicating that the underpinning of pah1Δ mutant defects was the excess synthesis of membrane phospholipids.

Rational design of organelle compartments in cells

Rational design of organelle compartments in cells

The endocannabinoid 2-arachidonoylglicerol decreases calcium induced cytochrome c release from liver mitochondria

2-Arachidonoylglicerol (2-AG) is an endocannabinoid that mimics the pharmacological effects of Δ⁹ tetrahydrocannabinol, the psychoactive component of the plant Cannabis sativa. It is present in many mammalian tissues, such as brain, liver, spleen, heart and kidney, where it exerts different biological effects either receptor mediated or independently of receptor activation. This work analyzes the effects of 2-AG on liver mitochondrial functions. It is shown that 2-AG causes a relevant decrease of calcium induced cyclosporine A sensitive cytochrome c release from mitochondria, a process representing an early event of the apoptotic program. Cyclosporin sensitive matrix swelling and ROS production measured under the same conditions are, on the contrary, almost unaffected or even enhanced, respectively, by 2-AG. Furthemore, 2-AG is found to stimulate resting state succinate oxidase activity and to inhibit oligomycin sensitive F₀F₁ ATP synthase activity. All these effects are apparently associated with 2-AG dependent alteration in the fluidity of the mitochondrial membranes, which was measured as generalized polarization of laurdan fluorescence.

Mitochondria-Dependent Reactive Oxygen Species-Mediated Programmed Cell Death Induced by 3,3′-Diindolylmethane through Inhibition of F0F1-ATP Synthase in Unicellular Protozoan ParasiteLeishmania donovani

Mitochondria are the principal site for the generation of cellular ATP by oxidative phosphorylation. F0F1-ATP synthase, a complex V of the electron transport chain, is an important constituent of mitochondria-dependent signaling pathways involved in apoptosis. In the present study, we have shown for the first time that 3,3'-diindolylmethane (DIM), a DNA topoisomerase I poison, inhibits mitochondrial F0F1-ATP synthase of Leishmania donovani and induces programmed cell death (PCD), which is a novel insight into the mechanism in protozoan parasites. DIM-induced inhibition of F0F1-ATP synthase activity causes depletion of mitochondrial ATP levels and significant stimulation of mitochondrial reactive oxygen species (ROS) production, followed by depolarization of mitochondrial membrane potential (DeltaPsi(m)). Because DeltaPsi(m) is the driving force for mitochondrial ATP synthesis, loss of DeltaPsi(m) results in depletion of cellular ATP level. The loss of DeltaPsi(m) causes the cellular ROS generation and in turn leads to the oxidative DNA lesions followed by DNA fragmentation. In contrast, loss of DeltaPsi(m) leads to release of cytochrome c into the cytosol and subsequently activates the caspase-like proteases, which lead to oligonucleosomal DNA cleavage. We have also shown that mitochondrial DNA-depleted cells are insensitive to DIM to induce PCD. Therefore, mitochondria are necessary for cytotoxicity of DIM in kinetoplastid parasites. Taken together, our study indicates for the first time that DIM-induced mitochondrial dysfunction by inhibition of F0F1-ATP synthase activity leads to PCD in Leishmania spp. parasites, which could be exploited to develop newer potential therapeutic targets.

Downmodulation of mitochondrial F0F1ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F(0)F(1) ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF(1). We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F(1) sector of F(0)F(1) ATP synthase by promoting IF(1) binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF(1) binding (basal ATPase activity) or detaching bound IF(1) at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 muM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF(1) from F(0)F(1) ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF(1)-stripping conditions, indicating that diazoxide alters alkaline IF(1) release. Diazoxide inhibition of ATPase activity in IF(1)-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF(1) to F(0)F(1) ATP synthase and enhances IF(1) binding indirectly by mildly uncoupling and depolarizing mitochondria.

F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat.

The amplitude of coronary reactive hyperemia (CRH), elicited by 15 s of ischemia, is reduced in hearts subjected to 5 min of ischemic preconditioning (IP). F0F1 ATP synthase activity and ATP concentration are also altered by IP. We hypothesized that F0F1 ATP synthase is differently modulated by the inhibitor protein IF(1) during CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were obtained before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, after IP (Cprec), during CRHprec, and 4 min after CRHprec. F0F1 ATP synthase activity, ATP concentration, and ATP-to-ADP ratio (ATP/ADP) were determined. Compared with CRHnp, IP blunted CRHprec. F0F1 ATP synthase activity transiently increased during CRHnp, decreased 4 min after CRHnp, and returned to control 2 min later; it was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by IF1. During CRHnp, ATP concentration and ATP/ADP were reduced compared with Cnp and began to rise 6 min thereafter. During Cprec, both parameters were transiently reduced but increased during and after CRHprec. Hence, during CRHnp, F0F1 ATP synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why a few seconds of occlusion do not induce IP. After IP, F0F1 ATP synthase activity is blunted, and it is not affected by a subsequent 15 s of occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F0F1 ATP synthase activity may be a feature of the preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of reduced ATP hydrolysis by cytoplasmic ATPases.

Dose-dependent inhibition of mitochondrial ATP synthase by 17β-estradiol

Mitochondria produce energy through oxidative phosphorylation. A key enzyme in this pathway is F0F1-ATP synthase, catalyzing ATP production from ADP and inorganic phosphate. Recently a subunit of F0F1-ATP synthase, oligomycin sensitivity-conferring protein, was identified as a new estradiol-binding protein. Estradiol could directly modulate mitochondrial ATP synthase activity through this subunit. In addition, intracellular ATP levels play a role in apoptotic death, which is an energy-dependent process requiring functioning mitochondria. Here we examined the effect of 17β -estradiol on F0F1-ATP synthase directly (in permeabilized cells) and in intact osteoclastic FLG 29.1 cells, a model of inducible apoptosis. The baseline F0F1-ATP synthase activity of FLG 29.1 cells was 4.485 nmol/min per mg. Estradiol rapidly inhibited F0F1-ATP synthase activity in the physiological range (half-inhibition concentration, IC50, of 30 nmol/l). With 1 nmol/l of estradiol, the inhibition was already significant (8-10% inhibition, p < 0.01) and with 100 nmol/l residual enzyme activity was only 15% (85% inhibition, p < 0.01). In addition, the effect of estradiol appeared to be directed towards F0F1-ATP synthase, since succinate-sustained respiration, uncoupled from the electron transport chain, was unaffected by estradiol. We assayed F0F1-ATP synthase activity in FLG 29.1 cells during inducible apoptosis. No significant difference of ATP synthesis was detected in apoptotic cells versus controls. In conclusion, we showed a new non-genomic effect of estradiol on a key mitochondrial enzyme, which thereby directly modulates cellular energy metabolism.

Dose-dependent inhibition of mitochondrial ATP synthase by 17β-estradiol

Mitochondria produce energy through oxidative phosphorylation. A key enzyme in this pathway is F0F1-ATP synthase, catalyzing ATP production from ADP and inorganic phosphate. Recentlya subunit of F0F1-ATP synthase, oligomycin sensitivity-conferring protein, was identified as a new estradiol-binding protein. Estradiol could directly modulate mitochondrial ATP synthaseactivity through this subunit. In addition, intracellular ATP levels play a role in apoptotic death, which is an energy-dependent process requiring functioning mitochondria. Here we examined the effectof 17β -estradiol on F0F1-ATP synthase directly (in permeabilized cells) and in intact osteoclastic FLG 29.1 cells, a model of inducible apoptosis. The baseline F0F1-ATPsynthase activity of FLG 29.1 cells was 4.485 nmol/min per mg. Estradiol rapidly inhibited F0F1-ATP synthase activity in the physiological range (half-inhibition concentration, IC50,of 30 nmol/l). With 1 nmol/l of estradiol, the inhibition was already significant (8-10% inhibition, p < 0.01) and with 100 nmol/l residual enzyme activity was only 15% (85% inhibition,p < 0.01). In addition, the effect of estradiol appeared to be directed towards F0F1-ATP synthase, since succinate-sustained respiration, uncoupled from the electron transport chain,was unaffected by estradiol. We assayed F0F1-ATP synthase activity in FLG 29.1 cells during inducible apoptosis. No significant difference of ATP synthesis was detected in apoptoticcells versus controls. In conclusion, we showed a new non-genomic effect of estradiol on a key mitochondrial enzyme, which thereby directly modulates cellular energy metabolism.

Lengthening the Second Stalk of F1F0 ATP Synthase in Escherichia coli

In Escherichia coli F(1)F(0) ATP synthase, the two b subunits dimerize forming the peripheral second stalk linking the membrane F(0) sector to F(1). Previously, we have demonstrated that the enzyme could accommodate relatively large deletions in the b subunits while retaining function (Sorgen, P. L., Caviston, T. L., Perry, R. C., and Cain, B. D. (1998) J. Biol. Chem. 273, 27873-27878). The manipulations of b subunit length have been extended by construction of insertion mutations into the uncF(b) gene adding amino acids to the second stalk. Mutants with insertions of seven amino acids were essentially identical to wild type strains, and mutants with insertions of up to 14 amino acids retained biologically significant levels of activity. Membranes prepared from these strains had readily detectable levels of F(1)F(0)-ATPase activity and proton pumping activity. However, the larger insertions resulted in decreasing levels of activity, and immunoblot analysis indicated that these reductions in activity correlated with reduced levels of b subunit in the membranes. Addition of 18 amino acids was sufficient to result in the loss of F(1)F(0) ATP synthase function. Assuming the predicted alpha-helical structure for this area of the b subunit, the 14-amino acid insertion would result in the addition of enough material to lengthen the b subunit by as much as 20 A. The results of both insertion and deletion experiments support a model in which the second stalk is a flexible feature of the enzyme rather than a rigid rod-like structure.

Decrease of cytochrome c oxidase protein in heart mitochondria of copper-deficient rats.

Copper deficiency has been reported to be associated with decreased cytochrome c oxidase activity, which in turn may be responsible for the observed mitochondrial impairment and cardiac failure. We isolated mitochondria from hearts of copper-deficient rats: cytochrome c oxidase activity was found to be lower than in copper-adequate mitochondria. The residual activity paralleled copper content of mitochondria and also corresponded with the heme amount associated with cytochrome aa3. In fact, lower absorption in the alpha-band region of cytochrome aa3 was found for copper-deficient rat heart mitochondria. Gel electrophoresis of protein extracted from mitochondrial membranes allowed measurements of protein content of the complexes of oxidative phosphorylation, revealing a lower content of complex IV protein in copper-deficient rat heart mitochondria. The alterations caused by copper deficiency appear to be specific for cytochrome c oxidase. Changes were not observed for F0F1ATP synthase activity, for heme contents of cytochrome c and b, and for protein contents of complexes I, III and V. The present study demonstrates that the alteration of cytochrome c oxidase activity observed in copper deficiency is due to a diminished content of assembled protein and that shortness of copper impairs heme insertion into cytochrome c oxidase.