ATP synthesis

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F H F I and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F H F I . The proton transfer through F H F I after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATPsynthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.

Abamectin (ABA) is a macrocyclic lactone of the avermectin family used worldwide as an antiparasitic agent in farm animals and pets and as the active ingredient of insecticides and nematicides. In this study, the effects of abamectin on the bioenergetics of mitochondria isolated from rat liver were evaluated. Mitochondria are responsible for converting the energy released by electron transport and stored as the binding energy molecule ATP. Xenobiotics that interfere with its synthesis or utilization can be acutely or chronically toxic. Abamectin (5-25 lM) caused concentration-dependent inhibition of the respiratory chain without affecting the membrane potential or the activity of enzymes NADH dehydrogenase or succinate dehydrogenase. This behavior is similar to oligomycin and carboxyatractyloside and suggests direct action on F o F 1 -ATPase and/or the adenine nucleotide translocator (ANT). ABA more pronouncedly inhibited ATPase phosphohydrolase activity in intact, uncoupled mitochondria than in freeze-thawed disrupted mitochondria. ADP-stimulated depolarization of the mitochondrial membrane potential was also inhibited by ABA. Our results indicate that ABA interacts more specifically with the ANT, resulting in functional inhibition of the translocator with consequent impairment of mitochondrial bioenergetics. This effect could be involved in the ABA toxicity to hepatocytes.

The in vitro interference of some of most important nonsteroidal anti-inflammatory drugs (NSAIDs) with the respiration of rat kidney (renal cortex) mitochondria and ATP synthesis was evaluated. Acetylsalicylic Nonsteroidal anti-inflammatory drugs (NSAIDs, 3 for acid, diclofenac sodium, mefenamic acid, and piroxia review see Ref. (1)) are a group of agents with anticam both uncoupled and inhibited oxidative phosinflammatory, analgesic, antipyretic, and platelet-inphorylation in mitochondria energized with glutahibitory actions. They are some of the most widely used mate plus malate or with succinate, while dipyrone therapeutic agents in clinical practice at present, and only uncoupled and paracetamol only inhibited it. The additional indications for them are rapidly increasing. drug concentrations affecting mitochondrial respiration were in the low to middle micromolar range for Hence, from a public health perspective, their putative diclofenac, mefenamic acid, and piroxicam, and in the cytotoxicity in terms of energy metabolism should be low millimolar range for acetylsalicylic acid, dipyrone, determined because of their widespread availability and paracetamol. The pattern of inhibition, except for and use (2). the paracetamol, was similar to that expressed by the The gastrointestinal side effects of NSAIDs have atrespiratory chain inhibitors. NSAIDs also inhibited tracted most attention, and it has been suggested that the rate of ATP synthesis in mitochondria energized the first stage of the pathogenic framework for NSAIDwith glutamate plus malate, as well as the phosphoryinduced enteropathy represents specific biochemical allation potential of mitochondria. The IC 50 values for terations initiated by the uncoupling of oxidative phosrate of ATP synthesis, using 2 mM ADP, were about 0.1 phorylation in mitochondria (3). It has become increas-mM for diclofenac sodium and mefenamic acid, 0.7 mM ingly apparent that the kidney is also an important for piroxicam, and in the range of 5-8 mM for acetylsaltarget for the untoward effects of NSAIDs (2, 4-7). icylic acid, dipyrone, and paracetamol. The potential These agents have been known to produce a wide array for renal energetic cytotoxicity of NSAIDs is discussed of untoward renal effects that may be divided into sevconsidering their ability to interact with the oxidative eral distinct nephrologic syndromes, including acute phosphorylation in rat renal cortex mitochondria. A renal failure, chronic renal injury, nephrotic syndrome comparison is made with the interference of salicylate, and interstitial nephritis, abnormalities of water methe main metabolite of acetylsalicylic acid, and a clastabolism, and abnormalities of sodium and potassium sical uncoupler of oxidative phosphorylation. ᭧ 1996 homeostasis (2, 4).

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of seventeen bioflavonoid compounds on purified F 1 or membrane bound F 1 F O E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC 50 ~0.07mM) > silymarin (IC 50 ~0.11mM) > baicalein (IC 50 ~0.29mM) > silibinin (IC 50 ~0.34mM) > rimantadine (IC 50 ~2.0mM) > amantidin (IC 50 ~2.5mM) > epicatechin (IC 50 ~4.0mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F 1 F o membrane preparations isolated purified F 1 . ATPase and growth assays suggested that the bioflavonoids compounds used in this study inhibited F 1 -ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-␤-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the ␥-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the ␥-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-␤-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-␤-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC 50 ∼14 M) followed by quercetin (IC 50 ∼33 M), quercetin-3-␤-d glucoside (IC 50 ∼71 M), resveratrol (IC 50 ∼94 M), quercitrin (IC 50 ∼120 M). Inhibition was identical in both F 1 F o membrane preparations as well as in isolated purified F 1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-␤-d glucoside inhibited only ATPase activity and not ATP synthesis.

The assessment of mitochondrial properties in skeletal muscle is important in clinical research, for instance in the study of diabetes. The gold standard to measure mitochondrial capacity non-invasively is the phosphocreatine (PCr) recovery rate after exercise, measured by 31 P Magnetic Resonance spectroscopy (31 P MRS). Here, we sought to expand the evidence base for an alternative method to assess mitochondrial properties which uses 31 P MRS measurement of the Pi content of an alkaline compartment attributed to mitochondria (Pi 2 ; as opposed to cytosolic Pi (Pi 1)) in resting muscle at high magnetic field. Specifically, the PCr recovery rate in human quadriceps muscle was compared with the signal intensity of the Pi 2 peak in subjects with varying mitochondrial content of the quadriceps muscle as a result of athletic training, and the results were entered into a mechanistic computational model of mitochondrial metabolism in muscle to test if the empirical relation between Pi 2 /Pi 1 ratio and the PCr recovery was consistent with theory. Localized 31 P spectra were obtained at 7T from resting vastus lateralis muscle to measure the intensity of the Pi 2 peak. In the endurance trained athletes a Pi 2 /Pi 1 ratio of 0.07 ± 0.01 was found, compared to a significantly lower (p<0.05) Pi 2 /Pi 1 ratio of 0.03 ± 0.01 in the normally active group. Next, PCr recovery kinetics after in magnet bicycle exercise were measured at 1.5T. For the endurance trained athletes, a time constant τ PCr 12 ± 3 s was found, compared to 24 ± 5s in normally active subjects. Without any parameter optimization the computational model prediction matched the experimental data well (r 2 of 0.75). Taken together, these results suggest that the Pi 2 resonance in resting human skeletal muscle observed at 7T provides a quantitative MR-based functional measure of mitochondrial density.

The interactions of zinc pyrithione (ZnPT) with rat liver mitochondria were investigated. Since most of the organometals, principally the triorganotin compounds, induce the inhibition of ATP synthesis in rat liver mitochondria, the efficiency of the ATP synthesis was measured in the presence of ZnPT. The results indicate that ZnPT inhibits ATP synthesis. In order to individuate the molecular mechanism responsible for a failure in ATP synthesis, all of the steps involved in ATP synthesis or in its inhibition were investigated separately, i.e. the respiratory chain, the uncoupling effect, the ATPase and the opening of a permeability pore. All of the steps are inhibited by ZnPT, but the crucial one, the one responsible for the inhibition of ATP synthesis, seems to be the opening of a small-size cyclosporine-sensitive pore. The results are different from those obtained using other organometallic compounds, but are similar to those obtained when using methylmercury and Zn 2+ , both of which also induce the opening of a cyclosporine-sensitive pore. However, although Hg 2+ and Zn 2+ would seem to induce the opening of large-size pores, in the case of ZnPT the pores involved are of a small size. This action mechanism seems to exclude the possibility that ZnPT is a deliverer of Zn 2+ .

The process of cellular respiration occurs for energy production through catabolic reactions, generally with glucose as the first process step. In the present work, we introduce a novel concept for understanding this process, based on our conclusion that glucose metabolism is coupled to the pentose phosphate pathway (PPP) and extra-mitochondrial oxidative phosphorylation in a closed-loop process. According to the current standard model of glycolysis, glucose is first converted to glucose 6-phosphate (glucose 6-P) and then to fructose 6-phosphate, glyceraldehyde 3-phosphate and pyruvate, which then enters the Krebs cycle in the mitochondria. However, it is more likely that the pyruvate will be converted to lactate. In the PPP, glucose 6-P is branched off from glycolysis and used to produce NADPH and ribulose 5-phosphate (ribulose 5-P). Ribulose 5-P can be converted to fructose 6-P and glyceraldehyde 3-P. In our view, a circular process can take place in which the ribulose 5-P produced by the PPP enters the glycolysis pathway and is then retrogradely converted to glucose 6-P. This process is repeated several times until the complete degradation of glucose 6-P. The role of mitochondria in this process is to degrade lipids by beta-oxidation and produce acetyl-CoA; the function of producing ATP appears to be only secondary. This proposed new concept of cellular bioenergetics allows the resolution of some previously unresolved controversies related to cellular respiration and provides a deeper understanding of metabolic processes in the cell, including new insights into the Warburg effect.

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of seventeen bioflavonoid compounds on purified F 1 or membrane bound F 1 F O E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC 50 ~0.07mM) > silymarin (IC 50 ~0.11mM) > baicalein (IC 50~0 .29mM) > silibinin (IC 50 ~0.34mM) > rimantadine (IC 50 ~2.0mM) > amantidin (IC 50 ~2.5mM) > epicatechin (IC 50 4.0mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F 1 F o membrane preparations isolated purified F 1. ATPase and growth assays suggested that the bioflavonoids compounds used in this study inhibited F 1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

The effect of the addition of KCI, at constant osmolarity, was investigated on oxidative phosphorylation in isolated yeast mitochondria. KCI stimulated both respiration and ATP synthesis rates without changing the ATP/O ratio. KC1 did not change the relationships between respiration rates and the protonmotive force. Since the K+/H + exchange activity was active under these conditions, the stimulatory effect of respiration could be explained by the net proton entry caused by the electrophoretic K + entry/electroneutral K+/H ÷ exchange cycle. On the other hand, K ÷ entry stimulated phosphate accumulation and transport under non-phosphorylating conditions and decreased the kinetic control by phosphate transport under phosphorylating conditions. Additionally, the stimulation of ATP synthesis strongly depended on the activity of phosphate transport. Taken together, these data showed that electrophoretic K+-entry and electroneutral K+/H + exchange occurred in phosphorylating yeast mitochondria but did not promote any uncoupling between respiration and ATP synthesis.

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of seventeen bioflavonoid compounds on purified F 1 or membrane bound F 1 F O E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC 50 ~0.07mM) > silymarin (IC 50 ~0.11mM) > baicalein (IC 50~0 .29mM) > silibinin (IC 50 ~0.34mM) > rimantadine (IC 50 ~2.0mM) > amantidin (IC 50 ~2.5mM) > epicatechin (IC 50 4.0mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F 1 F o membrane preparations isolated purified F 1. ATPase and growth assays suggested that the bioflavonoids compounds used in this study inhibited F 1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

(1) EDTA-resolved particles (partially depleted of CF I) from lettuce chloroplast thylakoids were reconstituted with CF I from lettuce and with TF~ from the thermophilic bacterium PS3. The binding affinity of both F I proteins for the membranal CF 0 is similar. (2) The phosphorylation activities of the EDTA-resoived particles after reconstitution with either the tentoxin-sensitive CF 1 or the tentoxin-resistant TFt are equally inhibited by tentoxin. The same degree of reconstitution of photophosphorylation is observed either with the native TFI, the DCCD-modified TF I or the DCCD-modified CF 1. Particles reconstituted with native CF I always catalyzed a higher rate of ATP formation. (3) NaBr-resolved particles (CFt-depleted) bind TF I with the reconstitution of the light-dependent 14 ÷ uptake. 14owever, no significant enhancement of ATP formation accompanies the binding of TF ! to the membranal CF 0. (4) These findings suggest a close relationship between the F0-F I complex of the prokaryotic thermophilic bacterium PS3 and that from lettuce, a eukaryotic organism. It is also apparent that the rebinding of added TF I or CF I to CFt-depleted particles reforms ATP synthetase complexes that are not fully catalytically functional. In particular, the TF~ incorporated into the membrane appears to have only a structural role of sealing the membrane and preventing protons from leaking out but does not participate in the catalytic process itself. The endogenous CFI that is present in the native undissociated ATP synthetase complex and still remains in these CFi-partially depleted membranes is the catalytically active enzyme species.

Waltheria brachypetala Turcz., owns special structural features as two α,β-unsaturated carbonyl groups and a side alkyl chain that can compete with the quinones involved in the pool of plastoquinones at photosystem II (PSII). In this work, we showed that the alkaloid is an inhibitor of Hill reaction and its target was located at the acceptor side of PSII. Studies of chlorophyll (Chl) a fluorescence showed a J-band that indicates direct action of antidesmone in accumulation of Q A − (reduced plastoquinone A) due to the electron transport blocked at the Q B (plastoquinone B) level similar to DCMU. In vivo assays indicated that antidesmone is a selective post-emergent herbicide probe at 300 μM by reducing the biomass production of Physalis ixacarpa plants. Furthermore, antidesmone also behaves as pre-emergent herbicide due to inhibit Physalis ixacarpa plant growth about 60%. Antidesmone, a natural product containing a 4(1H)-pyridones scaffold, will serve as a valuable tool in further development of a new class of herbicides.

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-␤-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the ␥-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the ␥-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-␤-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-␤-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC 50 ∼14 M) followed by quercetin (IC 50 ∼33 M), quercetin-3-␤-d glucoside (IC 50 ∼71 M), resveratrol (IC 50 ∼94 M), quercitrin (IC 50 ∼120 M). Inhibition was identical in both F 1 F o membrane preparations as well as in isolated purified F 1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-␤-d glucoside inhibited only ATPase activity and not ATP synthesis.

Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming}, EC 6.3.4.3) from Clos~ridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K ÷ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrofolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate.

Expcrimcnls with resting cells oT Ace/ohacroirrr,r ~~oc/ii wcrc pcrl'ormcd IO clucidatc the coupling ion used by the ATP synihasc. A. woudii synlhcsizcd ATP in rcsponsc to an artilicial dpH. indicating the prcscncc ol'a proton-translocaling ATPasc. On the o:hcr lrdnd. a dpNa. as well as a proton diffusion potcnlial. could scrvc as a driving force for ATP synthesis with the lallcr strictly dcpcndcnl on Na-. Thcsc resuhs arc indicative Tar ~hc prcscncc of a Na'-translocuting ATP syn~hasc in A. ~oo~lii.

Many cancer cells are characterized by high rate of glycolysis and reduced rate of aerobic respiration, whose mechanism is still elusive. Here we investigate the down-regulation of oxidative phosphorylation (OXPHOS) in K-ras transformed mouse fibroblasts as compared to a control counterpart. Transcriptional analysis showed different expression levels of several OXPHOS nuclear genes in the two cell lines. In particular, during the exponential growth phase most genes encoding proteins of Complex I were expressed at lower levels in transformed cells. Consistently, a significant decrease of Complex I content was found in transformed cells. Moreover, analysis of NAD-dependent respiration and ATP synthesis indicated a strong decrease of Complex I activity in the mitochondria from neoplastic cells, that was confirmed by direct assay of the enzyme redox activity. At variance, succinate-dependent respiration and ATP synthesis were not significantly affected. Taken together, our results provide the new insight that the reduction of respiration observed in K-ras transformed cells is specifically due to a Complex I activity decrease.

There is considerable interest in improving plant productivity by altering the dynamic responses of photosynthesis in tune with natural conditions. This is exemplified by the ‘energy-dependent&#39; form of non-photochemical quenching ( q E ), the formation and decay of which can be considerably slower than natural light fluctuations, limiting photochemical yield. In addition, we recently reported that rapidly fluctuating light can produce field recombination-induced photodamage (FRIP), where large spikes in electric field across the thylakoid membrane (Δ ψ ) induce photosystem II recombination reactions that produce damaging singlet oxygen ( 1 O 2 ). Both q E and FRIP are directly linked to the thylakoid proton motive force ( pmf ), and in particular, the slow kinetics of partitioning pmf into its ΔpH and Δ ψ components. Using a series of computational simulations, we explored the possibility of ‘hacking&#39; pmf partitioning as a target for improving photosynthesis. Under a range o...

This study evaluates the use of role-playing games (RPGs) as a methodological approach for teaching cellular biology, assessing student satisfaction, learning outcomes, and retention of acquired knowledge. First-year undergraduate medical students at two Brazilian public universities attended either an RPG-based class (RPG group) or a lecture (lecture-based group) on topics related to cellular biology. Pre- and post-RPG-based class questionnaires were compared to scores in regular exams and in an unannounced test one year later to assess students&#39; attitudes and learning. From the 230 students that attended the RPG classes, 78.4% responded that the RPG-based classes were an effective tool for learning; 55.4% thought that such classes were better than lectures but did not replace them; and 81% responded that they would use this method. The lecture-based group achieved a higher grade in 1 of 14 regular exam questions. In the medium-term evaluation (one year later), the RPG group sc...

Waltheria brachypetala Turcz., owns special structural features as two α,β-unsaturated carbonyl groups and a side alkyl chain that can compete with the quinones involved in the pool of plastoquinones at photosystem II (PSII). In this work, we showed that the alkaloid is an inhibitor of Hill reaction and its target was located at the acceptor side of PSII. Studies of chlorophyll (Chl) a fluorescence showed a J-band that indicates direct action of antidesmone in accumulation of Q A − (reduced plastoquinone A) due to the electron transport blocked at the Q B (plastoquinone B) level similar to DCMU. In vivo assays indicated that antidesmone is a selective post-emergent herbicide probe at 300 μM by reducing the biomass production of Physalis ixacarpa plants. Furthermore, antidesmone also behaves as pre-emergent herbicide due to inhibit Physalis ixacarpa plant growth about 60%. Antidesmone, a natural product containing a 4(1H)-pyridones scaffold, will serve as a valuable tool in further development of a new class of herbicides.

Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its ρ0 counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate lev...

(1) EDTA-resolved particles (partially depleted of CF I) from lettuce chloroplast thylakoids were reconstituted with CF I from lettuce and with TF~ from the thermophilic bacterium PS3. The binding affinity of both F I proteins for the membranal CF 0 is similar. (2) The phosphorylation activities of the EDTA-resoived particles after reconstitution with either the tentoxin-sensitive CF 1 or the tentoxin-resistant TFt are equally inhibited by tentoxin. The same degree of reconstitution of photophosphorylation is observed either with the native TFI, the DCCD-modified TF I or the DCCD-modified CF 1. Particles reconstituted with native CF I always catalyzed a higher rate of ATP formation. (3) NaBr-resolved particles (CFt-depleted) bind TF I with the reconstitution of the light-dependent 14 ÷ uptake. 14owever, no significant enhancement of ATP formation accompanies the binding of TF ! to the membranal CF 0. (4) These findings suggest a close relationship between the F0-F I complex of the prokaryotic thermophilic bacterium PS3 and that from lettuce, a eukaryotic organism. It is also apparent that the rebinding of added TF I or CF I to CFt-depleted particles reforms ATP synthetase complexes that are not fully catalytically functional. In particular, the TF~ incorporated into the membrane appears to have only a structural role of sealing the membrane and preventing protons from leaking out but does not participate in the catalytic process itself. The endogenous CFI that is present in the native undissociated ATP synthetase complex and still remains in these CFi-partially depleted membranes is the catalytically active enzyme species.

The aim of this study was to determine if the dietary benefits of bioflavonoids are linked to the inhibition of ATP synthase. We studied the inhibitory effect of seventeen bioflavonoid compounds on purified F 1 or membrane bound F 1 F O E. coli ATP synthase. We found that the extent of inhibition by bioflavonoid compounds was variable. Morin, silymarin, baicalein, silibinin, rimantadin, amantidin, or, epicatechin resulted in complete inhibition. The most potent inhibitors on molar scale were morin (IC 50 ~0.07mM) > silymarin (IC 50 ~0.11mM) > baicalein (IC 50~0 .29mM) > silibinin (IC 50 ~0.34mM) > rimantadine (IC 50 ~2.0mM) > amantidin (IC 50 ~2.5mM) > epicatechin (IC 50 4.0mM). Inhibition by hesperidin, chrysin, kaempferol, diosmin, apigenin, genistein, or rutin was partial in the range of 40-60% and inhibition by galangin, daidzein, or luteolin was insignificant. The main skeleton, size, shape, geometry, and position of functional groups on inhibitors played important role in the effective inhibition of ATP synthase. In all cases inhibition was found fully reversible and identical in both F 1 F o membrane preparations isolated purified F 1. ATPase and growth assays suggested that the bioflavonoids compounds used in this study inhibited F 1-ATPase as well as ATP synthesis nearly equally, which signifies a link between the beneficial effects of dietary bioflavonoids and their inhibitory action on ATP synthase.

We have studied the inhibitory effect of five polyphenols namely, resveratrol, piceatannol, quercetin, quercetrin, and quercetin-3-␤-d glucoside on Escherichia coli ATP synthase. Recently published X-ray crystal structures of bovine mitochondrial ATP synthase inhibited by resveratrol, piceatannol, and quercetin, suggest that these compounds bind in a hydrophobic pocket between the ␥-subunit C-terminal tip and the hydrophobic inside of the surrounding annulus in a region critical for rotation of the ␥-subunit. Herein, we show that resveratrol, piceatannol, quercetin, quercetrin, or quercetin-3-␤-d glucoside all inhibit E. coli ATP synthase but to different degrees. Whereas piceatannol inhibited ATPase essentially completely (∼0 residual activity), inhibition by other compounds was partial with ∼20% residual activity by quercetin, ∼50% residual activity by quercetin-3-␤-d glucoside, and ∼60% residual activity by quercetrin or resveratrol. Piceatannol was the most potent inhibitor (IC 50 ∼14 M) followed by quercetin (IC 50 ∼33 M), quercetin-3-␤-d glucoside (IC 50 ∼71 M), resveratrol (IC 50 ∼94 M), quercitrin (IC 50 ∼120 M). Inhibition was identical in both F 1 F o membrane preparations as well as in isolated purified F 1. In all cases inhibition was reversible. Interestingly, resveratrol and piceatannol inhibited both ATPase and ATP synthesis whereas quercetin, quercetrin or quercetin-3-␤-d glucoside inhibited only ATPase activity and not ATP synthesis.

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its ρ0 counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate lev...

Stimulation of mitochondrial respiration by physiological concentrations of Ca2+ was studied to determine which components of oxidative phosphorylation are affected by Ca2+. The kinetic dependence of the respiratory chain, phosphorylation subsystem and proton leak on the mitochondrial membrane potential in isolated rat heart mitochondria respiring on 2-oxoglutarate or succinate was measured at two different concentrations of external free Ca2+. The results show that proton leak is not directly affected by Ca2+, but that both the respiratory and phosphorylation systems can be directly stimulated by Ca2+ depending on conditions. Although Ca2+ directly stimulates the phosphorylation system, this has relatively little effect on respiration rate with 2-oxoglutarate in States 3 and 4 because the subsystem has little control over respiration. However, in intermediate states, the phosphorylation system has greater control and Ca2+ stimulation of this system contributes substantially to the ...

Waltheria brachypetala Turcz., owns special structural features as two α,β-unsaturated carbonyl groups and a side alkyl chain that can compete with the quinones involved in the pool of plastoquinones at photosystem II (PSII). In this work, we showed that the alkaloid is an inhibitor of Hill reaction and its target was located at the acceptor side of PSII. Studies of chlorophyll (Chl) a fluorescence showed a J-band that indicates direct action of antidesmone in accumulation of Q A − (reduced plastoquinone A) due to the electron transport blocked at the Q B (plastoquinone B) level similar to DCMU. In vivo assays indicated that antidesmone is a selective post-emergent herbicide probe at 300 μM by reducing the biomass production of Physalis ixacarpa plants. Furthermore, antidesmone also behaves as pre-emergent herbicide due to inhibit Physalis ixacarpa plant growth about 60%. Antidesmone, a natural product containing a 4(1H)-pyridones scaffold, will serve as a valuable tool in further development of a new class of herbicides.

NSAIDs; kidney; mitochondria; oxidative phosphorylation; FoF1-ATPase; ATP; ATP synthesis. The in vitro interference of some of most important nonsteroidal anti-inflammatory drugs (NSAIDs) with the respiration of rat kidney (renal cortex) mitochondria and ATP synthesis was evaluated. Acetylsalicylic Nonsteroidal anti-inflammatory drugs (NSAIDs, 3 for acid, diclofenac sodium, mefenamic acid, and piroxia review see Ref. (1)) are a group of agents with anticam both uncoupled and inhibited oxidative phosinflammatory, analgesic, antipyretic, and platelet-inphorylation in mitochondria energized with glutahibitory actions. They are some of the most widely used mate plus malate or with succinate, while dipyrone therapeutic agents in clinical practice at present, and only uncoupled and paracetamol only inhibited it. The additional indications for them are rapidly increasing. drug concentrations affecting mitochondrial respiration were in the low to middle micromolar range for Hence, from a public health perspective, their putative diclofenac, mefenamic acid, and piroxicam, and in the cytotoxicity in terms of energy metabolism should be low millimolar range for acetylsalicylic acid, dipyrone, determined because of their widespread availability and paracetamol. The pattern of inhibition, except for and use (2). the paracetamol, was similar to that expressed by the The gastrointestinal side effects of NSAIDs have atrespiratory chain inhibitors. NSAIDs also inhibited tracted most attention, and it has been suggested that the rate of ATP synthesis in mitochondria energized the first stage of the pathogenic framework for NSAIDwith glutamate plus malate, as well as the phosphoryinduced enteropathy represents specific biochemical allation potential of mitochondria. The IC 50 values for terations initiated by the uncoupling of oxidative phosrate of ATP synthesis, using 2 mM ADP, were about 0.1 phorylation in mitochondria (3). It has become increas-mM for diclofenac sodium and mefenamic acid, 0.7 mM ingly apparent that the kidney is also an important for piroxicam, and in the range of 5-8 mM for acetylsaltarget for the untoward effects of NSAIDs (2, 4-7). icylic acid, dipyrone, and paracetamol. The potential These agents have been known to produce a wide array for renal energetic cytotoxicity of NSAIDs is discussed of untoward renal effects that may be divided into sevconsidering their ability to interact with the oxidative eral distinct nephrologic syndromes, including acute phosphorylation in rat renal cortex mitochondria. A renal failure, chronic renal injury, nephrotic syndrome comparison is made with the interference of salicylate, and interstitial nephritis, abnormalities of water methe main metabolite of acetylsalicylic acid, and a clastabolism, and abnormalities of sodium and potassium sical uncoupler of oxidative phosphorylation. ᭧ 1996 homeostasis (2, 4).

Many cancer cells are characterized by high rate of glycolysis and reduced rate of aerobic respiration, whose mechanism is still elusive. Here we investigate the down-regulation of oxidative phosphorylation (OXPHOS) in K-ras transformed mouse fibroblasts as compared to a control counterpart. Transcriptional analysis showed different expression levels of several OXPHOS nuclear genes in the two cell lines. In particular, during the exponential growth phase most genes encoding proteins of Complex I were expressed at lower levels in transformed cells. Consistently, a significant decrease of Complex I content was found in transformed cells. Moreover, analysis of NAD-dependent respiration and ATP synthesis indicated a strong decrease of Complex I activity in the mitochondria from neoplastic cells, that was confirmed by direct assay of the enzyme redox activity. At variance, succinate-dependent respiration and ATP synthesis were not significantly affected. Taken together, our results provide the new insight that the reduction of respiration observed in K-ras transformed cells is specifically due to a Complex I activity decrease.

Ž. Currently three conceptions on the mechanism of the ATP synthesis in mitochondria MT exist: chemical, chemiosmotic and conformational relaxation. But a quantitative interpretation of the proposed theories comes across the significant difficulties. In this work, the mechanical energy dissipation in mitochondria of lymphocytes in a steady state and swollen condition was studied by means of a Ž. Ž. Ž. registration, the biological mechanochemiemission BMCHE-the mechanoluminescence ML in the optical region 200-650 nm and Ž. Ž. the radioemission RE in the radio range 100 Hz to 0.1 MHz of the electromagnetic spectrum, with the subsequent processing of the results in a microcomputer system. The ATP concentration determination in mitochondria was performed by the bioluminescent method. The results obtained show that parameters of ML and RE main peaks of the mean power density are different for MT in the steady and swollen state. The coherence of the ML and RE signals in mitochondria is registered. In steady state mitochondria the ATP quantity was more than in swollen organelles by 5.2 = 10 y8 Mr10 6 cells. Our experimental results indicate that there is a correlation between a relaxation of tense energy and BMCHE process in an MT membrane. The phenomenological consideration of BMCHE mechanisms in the MT membrane energy transformation which is utilized for various processes of a living cell functioning is given. A model of BMCHE synthesis of ATP is suggested.

We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at aminoacid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.

Leber's hereditary optic neuropathy (LHON) was the first maternally inherited disease to be associated with point mutations in mitochondrial DNA and is now considered the most prevalent mitochondrial disorder. The pathology is characterized by selective loss of ganglion cells in the retina leading to central vision loss and optic atrophy, prevalently in young males. The pathogenic mtDNA point mutations for LHON affect complex I with the double effect of lowering the ATP synthesis driven by complex I substrates and increasing oxidative stress chronically. In this review, we first consider the biochemical changes associated with the proton-translocating NADH-quinone oxidoreductase of mitochondria in cybrid cells carrying the most common LHON mutations. However, the LHON cybrid bioenergetic dysfunction is essentially compensated under normal conditions, i.e. in glucose medium, but is unrevealed by stressful conditions such as growing cybrids in glucose free/galactose medium, which forces cells to rely only on respiratory chain for ATP synthesis. In fact, the second part of this review deals with the investigation of LHON cybrid death pathway in galactose medium. The parallel marked changes in antioxidant enzymes, during the time-course of galactose experiments, also reveal a relevant role played by oxidative stress. The LHON cybrid model sheds light on the complex interplay amongst the different levels of biochemical consequences deriving from complex I mutations in determining neurodegeneration in LHON, and suggests an unsuspected role of bioenergetics in shaping cell death pathways.

Many cancer cells are characterized by high rate of glycolysis and reduced rate of aerobic respiration, whose mechanism is still elusive. Here we investigate the down-regulation of oxidative phosphorylation (OXPHOS) in K-ras transformed mouse fibroblasts as compared to a control counterpart. Transcriptional analysis showed different expression levels of several OXPHOS nuclear genes in the two cell lines. In particular, during the exponential growth phase most genes encoding proteins of Complex I were expressed at lower levels in transformed cells. Consistently, a significant decrease of Complex I content was found in transformed cells. Moreover, analysis of NAD-dependent respiration and ATP synthesis indicated a strong decrease of Complex I activity in the mitochondria from neoplastic cells, that was confirmed by direct assay of the enzyme redox activity. At variance, succinate-dependent respiration and ATP synthesis were not significantly affected. Taken together, our results provide the new insight that the reduction of respiration observed in K-ras transformed cells is specifically due to a Complex I activity decrease.

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines. To test the hypothesis that Bim facilitates GC-induced apoptosis, we reduced BIM mRNA levels and Bim protein levels by RNA interference in highly GC-sensitive pre-B ALL cells. Reducing Bim proteins by either electroporation of synthetic small interfering RNA (siRNA) duplexes or lentivirusmediated stable expression of short hairpin RNA inhibited the activation of caspase-3 and increased cell viability following GC exposure. We also observed that the extent of GC resistance correlated with siRNA silencing potency. siRNA duplexes that reduced only BimEL or BimEL and BimL (but not BimS) exhibited less GC resistance than a potent siRNA that silenced all three major isoforms, implying that induction of all three Bim proteins contributes to cell death. Finally, the modulation of GC-induced apoptosis caused by Bim silencing was independent of Bcl-2 expression levels, negating the hypothesis that the ratio of Bim to Bcl-2 regulates apoptosis. These results offer evidence that the induction of Bim by GC is a required event for the complete apoptotic response in pre-B ALL cells. Glucocorticoids (GCs) 1 are steroid hormones that maintain physiological homeostasis. Synthetic GCs such as dexametha

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed ( 6 60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H gradient ( s 60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc 1 complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc 1 complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, npropyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.

Photosynthetic formation of inorganic pyrophosphate (PPi) in RhodospiriUum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PPm synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PPi synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PPi synthesis much less. Even at low rates of electron transport a significant rate of PPi synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPI synthesis is inhibited. It is shown that PP~ synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions.

The light-dependent production of ATP and reductants by the photosynthetic apparatus in vivo involves a series of electron and proton transfers. Consideration is given as to how electron fluxes through photosystem I (PSI), using absorption spectroscopy, and through photosystem II (PSII), using chlorophyll fluorescence analyses, can be estimated in vivo. Measurements of light-induced electrochromic shifts using absorption spectroscopy provide a means of analyzing the proton fluxes across the thylakoid membranes in vivo. Regulation of these electron and proton fluxes is required for the thylakoids to meet the fluctuating metabolic demands of the cell. Chloroplasts exhibit a wide and flexible range of mechanisms to regulate electron and proton fluxes that enable chloroplasts to match light use for ATP and reductant production with the prevailing metabolic requirements. Non-invasive probing of electron fluxes through PSI and PSII, and proton fluxes across the thylakoid membranes can provide insights into the operation of such regulatory processes in vivo.

Uptake of Ca2+ by rat brain mitochondria causes an inhibition of respiratory stimulation by ADP, and the inhibition is relieved upon Na+-induced release of Ca*+ from the mitochondria, in accordance with earlier reports. We show that simultaneous uptake of Ca2+ and Mn2+ results in no inhibition of ADPstimulated respiration, indicating that Mn2+ prevents the Ca2+-induced inhibition of ATP synthesis, without preventing Ca2+ accumulation in the mitochondria. The results are discussed in relation to a possible involvement of the mitochondrial ATPase-inhibitor protein in the observed effects of Ca2+ and Mn'+. A TP synthesis ADP-stimulated respiration Brain mitochondria &+-induced inhibition A TPase-inhibitor protein

Eukaryotic cells contain two organelles originally derived from endosymbiotic bacteria: mitochondria and plastids (only plants). In eukaryotes, (owner mitochondria and chloroplast) ATP synthase complex is located in the inner membrane of mitochondria, and thylakoids membrane of chloroplast. ATP synthesis utilization and provision of both ADP and Pi need to be fine – tuned for optimal ATP synthase activity. Mitochondria and chloroplast have their DNA. The vast majority of mitochondrial and plastid proteins are encoded in the nucleus, synthesized by cytosolic ribosomes and subsequently imported into the organelles via active protein transport systems.

A large number of new insights and further intricate details of the molecular mechanisms of ATP synthesis and muscle contraction have been offered from the perspective of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction. In this paper, a new systems thermodynamics analysis of oxidative-and photo-phosphorylation has been performed. New experimental data has been reported on the inhibition of ATP synthesis by known specific anion channel blockers: the triorganotin compound, tributyltin chloride (TBTCl), and the stilbene compound 4,4 0-diisothiocyanostilbene-2,2 0-disulfonate (DIDS), and interpreted as supporting the new framework. A bioinformatic analysis of the interacting a-c regions in F O has been carried out to locate the anion binding pocket in the anion-proton symsequenceporter and insights into the coordination chemistry of the bound chloride in its internal cavity at the lipid-water interface have been obtained. The need to look at ATP synthesis in F O as a multisubstrate reaction has been emphasized and a detailed microscopic explanation of the mechanism of inhibition by these blockers and its relationship to the conformational cycle within F O has been postulated. Such detailed explanations of the role of membrane elements in a lipophilic region have been shown to lead to deeper understanding and to offer a more realistic and complete picture of biological energy transduction than considering the bilayer as ''mere insulation'', as in chemiosmotic dogma. Details of the elementary force production events by myosin II have been provided within the novel molecular systems framework of the RUT mechanism in which the S-1, the S-1-S-2 hinge, and the S-2 coiled coil all have essential roles in the in vivo contractile process. The new paradigm is shown to be consistent with the great body of experiments usually considered to support the swinging crossbridge/lever arm models of muscle contraction, because these events also occur during the cycle but as events preliminary to the occurrence of the power stroke. The crucial role of energy storage in mechanically strained nonequilibrium conformational states of myosin, specifically as a high-energy state of S-2 with uncoiled first few N-terminal heptads is highlighted. The propensity of these heptads to recoil and regain the resting state of the coiled coil is postulated to be a primary driving force of the power stroke. The new paradigms of ATP synthesis/muscle contraction have been shown to remove the inconsistencies present in previous theories and to have the sound backing of the first and second laws of thermodynamics, the principle of electrical neutrality, the laws of Newtonian mechanics, and the great conservation laws of mechanics. A systems integration of muscle contraction has been successfully made and a systems electrical analog constructed. The unifying laws and principles in the new theories have been further applied to understand the functioning of other protein molecular machines such as kinesin, ncd and unconventional myosins. Finally, the major physical, chemical, biological and technological implications arising as a result of this research have been discussed. Taken together, the new paradigms have been shown to solve fundamental molecular mechanistic problems in bioenergetics and motility and to offer a most detailed, unified and appealing picture of energy generation, transduction, storage and utilization processes in systems of biological molecular machines.

A large number of new insights and further intricate details of the molecular mechanisms of ATP synthesis and muscle contraction have been offered from the perspective of the torsional mechanism of energy transduction and ATP synthesis and the rotation-uncoiling-tilt (RUT) energy ...

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F 1 F 0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F 1 F 0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.

Gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from 5-week-old cold-acclimated and thermoneutral control ducklings. In vitro respiration (polarography) and ATP synthesis (bioluminescence) were determined at 25³C. Subsarcolemmal mitochondria showed a higher state 4 respiration and lower respiratory control and ADP/O ratio in cold-acclimated than in thermoneutral ducklings. Palmitate decreased the rate of ATP synthesis in both mitochondrial populations to about 30% of maximal but failed to abolish this process even at high concentrations. It is suggested that both expensive ATP synthesis and increased ATP hydrolysis could contribute synergistically to muscle non-shivering thermogenesis in cold-acclimated ducklings.

Gastrocnemius subsarcolemmal and intermyofibrillar mitochondria were isolated from 5-week-old cold-acclimated and thermoneutral control ducklings. In vitro respiration (polarography) and ATP synthesis (bioluminescence) were determined at 25° C. ...

Mitochondrial ATP synthase, a major ATP supplier in respiring cells, should be regulated in amount and in activity to respond to the varying demands of cells for ATP. We screened 80 protein kinase inhibitors and found that HeLa cells treated with four inhibitors exhibited reduced mitochondrial ATP synthesis activity. Consistently, knockdown of their target kinases (PKA, PKCd, CaMKII and smMLCK) resulted in a decrease in mitochondrial ATP synthesis activity. Among them, mitochondria of smMLCK-knockdown cells contained only a small amount of ATP synthase, while the aand bsubunits of ATP synthase were produced normally, suggesting that smMLCK affects assembly (or decay) of ATP synthase.

A new assay has been developed to measure mitochondrial ATP synthesis of cultured mammalian cells. Cells in a microplate are exposed to streptolysin O to make plasma membranes permeable without damaging mitochondrial function and ATP synthesis is monitored by luciferase. Addition of inhibitors of F o F 1-ATP synthase (F o F 1), respiratory chain, TCA cycle and ATP/ADP translocator, as well as knockdown of b-subunit of F o F 1 , resulted in loss of ATP synthesis. Compared with the conventional procedures that need mitochondria fractionation and detergent, this assay is simple, sensitive and suitable for highthroughput analysis of genes and drugs that could affect mitochondrial functional integrity as represented by ATP synthesis activity.