ATP Synthase Content Research Articles

Quercetin Reprograms Immunometabolism of Macrophages via the SIRT1/PGC-1α Signaling Pathway to Ameliorate Lipopolysaccharide-Induced Oxidative Damage.

The redox system is closely related to changes in cellular metabolism. Regulating immune cell metabolism and preventing abnormal activation by adding antioxidants may become an effective treatment for oxidative stress and inflammation-related diseases. Quercetin is a naturally sourced flavonoid with anti-inflammatory and antioxidant activities. However, whether quercetin can inhibit LPS-induced oxidative stress in inflammatory macrophages by affecting immunometabolism has been rarely reported. Therefore, the present study combined cell biology and molecular biology methods to investigate the antioxidant effect and mechanism of quercetin in LPS-induced inflammatory macrophages at the RNA and protein levels. Firstly, quercetin was found to attenuate the effect of LPS on macrophage proliferation and reduce LPS-induced cell proliferation and pseudopodia formation by inhibiting cell differentiation, as measured by cell activity and proliferation. Subsequently, through the detection of intracellular reactive oxygen species (ROS) levels, mRNA expression of pro-inflammatory factors and antioxidant enzyme activity, it was found that quercetin can improve the antioxidant enzyme activity of inflammatory macrophages and inhibit their ROS production and overexpression of inflammatory factors. In addition, the results of mitochondrial morphology and mitochondrial function assays showed that quercetin could upregulate the mitochondrial membrane potential, ATP production and ATP synthase content decrease induced by LPS, and reverse the mitochondrial morphology damage to a certain extent. Finally, Western blotting analysis demonstrated that quercetin significantly upregulated the protein expressions of SIRT1 and PGC-1α, that were inhibited by LPS. And the inhibitory effects of quercetin on LPS-induced ROS production in macrophages and the protective effects on mitochondrial morphology and membrane potential were significantly decreased by the addition of SIRT1 inhibitors. These results suggested that quercetin reprograms the mitochondria metabolism of macrophages through the SIRT1/PGC-1α signaling pathway, thereby exerting its effect of alleviating LPS-induced oxidative stress damage.

Cytochrome-c-oxidase and ATPsynthase content increases in the endometrium of the patients with adenomyosis.

Adenomyosis is characterized by overgrowth of endometrial glands and stroma in the myometrium and is associated with reduced apoptosis. One of the key participants in one of the pathways of apoptosis is cytochrome c, whose expression can be regulated by actin-binding proteins involved in the formation of structures that provide cell motility. The aim of the study was to determine the content of actin-binding proteins, cytochrome c, and terminal members of the mitochondrial respiratory chain in endometrial biopsies of patients with adenomyosis and the control group. The content of all studied proteins was determined by Western blotting, and the mRNA content of the corresponding genes was determined by quantitative RT-PCR. The relative content of alpha-actinin1 and mRNA of the gene encoding it in biopsy specimens from patients with adenomyosis was higher than in controls by 86 and 84% (p < 0.05), respectively. The relative content of alpha-actinin4 did not change, as did cytochrome c. The content of cytochrome-c-oxidase and ATPsynthase in the group with adenomyosis exceeded the control level by 270 and 121% (p < 0.05), respectively, but the relative content of mRNA of these genes did not change, which may indicate a change in regulation at the level of protein synthesis. The results may indicate a local increase in the synthesis of ATP in pathological endometrial cells, which indicates the possible effectiveness of local application of H+-ATP synthase inhibitors (for example, macrolide antibiotic) to reduce the severity of clinical symptoms of adenomyosis.

Изменения содержания АТФ-синтазы в нейронах мозга при экспериментальной церебральной ишемии

Introduction. ATP synthase is a key component of ATP synthesis. The study of its content in brain neurons in experimental cerebral ischemia may reflect changes in the functional state of different neurons and their sensitivity to the pathological effect. The study aimed to reveal patterns in ATP synthase immunoreactivity in neurons of various parts of the rat brain during subtotal ischemia of various durations. Materials and methods. Modeling of subtotal cerebral ischemia (SCI) was carried out by ligation of both common carotid arteries (n=12: control group – n=4, 30-minute SCI – n=4, 3-hour SCI – n=4); the control animals underwent a sham surgery. Frontal paraffin sections were stained according to the Nissl method to identify brain structures and immunohistochemically for ATP synthase. ATP synthase immunoreactivity was expressed in units of optical density ×103. Results. Changes in the immunoreactivity of ATP synthase in brain structures occur to varying degrees and not in all studied structures. The most pronounced and rapid decrease in the content of ATP synthase was observed in telencephalon, namely in the temporal and retrosplenial agranular cortex. The least pronounced changes in the cortex were in the structures of the hippocampus, in the neurons of layer II of the CA2 field. In the structures of the thalamus, hypothalamus, and midbrain the dynamic varied. In the structures of the medulla oblongata, the decrease in immunoreactivity occurs more slowly and less pronounced. Conclusion. During experimental cerebral ischemia the degree and rate of change in the content of ATP synthase in rat brain structures vary greatly and depend both on the brain region and the neurotransmitter nature of neurons. Keywords: ATP synthase, ischemia, immunohistochemistry, brain, rat

Correction with ω‑3 polyunsaturated fatty acids of disturbances in energy processes in the brain tissues of rats with subtotal cerebral ischemia

In the course of the work, the parameters of respiration of mitochondria of brain homogenates and the content of ATP synthase in rats with cerebral ischemia and under conditions of administration of ω-3 polyunsaturated fatty acids were studied. ω-3 polyunsaturated fatty acids have an anti-inflammatory effect due to their incorporation into the phospholipid layer of cell membranes of monocytes, leukocytes, endothelial cells, which is accompanied by a decrease in the production of inflammatory mediators and a decrease in leukocyte adhesion to the endothelial wall. Polyunsaturated fatty acids regulate vascular tone and prevent vascular vasoconstriction under the influence of catecholamines, which causes a moderate vasodilatory effect. ω-3 polyunsaturated fatty acids have a corrective effect on mitochondrial respiration, their beneficial effect on the structures of the hippocampus in conditions of subtotal cerebral ischemia and a lesser severity of manifestations of neurological deficit, compared with the group without their administration, are noted.

The content of ATP Synthase in Cerebral Ischemia of Varying Severity Comparative Analysis

Objective. Evaluation of changes in the content of ATP synthase in the parietal cortex and hippocampus of the brain of rats with ischemia of varying severity in a comparative aspect. Methods. The experiments were performed on 88 male outbred white rats weighing 260 ± 20 g. Brain ischemia was modeled under conditions of intravenous thiopental anesthesia (40-50 mg / kg). Total cerebral ischemia was modeled by decapitation of animals. The brain sampling was carried out 1 hour and 24 hours after decapitation - to study tissue respiration of mitochondria, as well as 1 hour later to determine the content of ATP synthase. Subtotal cerebral ischemia was modeled by simultaneous ligation of both common carotid arteries. The material was taken after 1 hour to determine the content of ATP synthase. Stepwise subtotal cerebral ischemia was performed by sequential ligation of both common carotid arteries with an interval of 7 days. The sampling was carried out 1 hour after ligation of the second common carotid artery in each of the subgroups. Partial cerebral ischemia was modeled by ligation of one common carotid artery on the right. The sampling was carried out 1 hour after the operation. Determination of the content of ATP synthase was carried out by immunohistochemical method using monoclonal antibodies. For this purpose, after decapitation, the brain was quickly removed from the rats, pieces of the cerebral cortex were fixed in zinc-ethanol-formaldehyde at + 4 ° C (overnight), then embeddedвinвparaffin. Results. In the group of stepwise subtotal cerebral ischemia, the smallest decrease in the content of ATP synthase was observed in the 1st subgroup with an interval between dressings of 7 days, while the greatest decrease in the content of the enzyme was noted in the 3rd subgroup with the minimum interval between the dressings of the common carotid artery (1 day). Modeling of more severe types of ischemic damage led to pronounced morphological changes in neurons in the parietal cortex and hippocampus of the rat brain - a decrease in their size, deformation of the perikarya, an increase in the degree of neuronal chromatophilia with their simultaneous wrinkling and subsequent death. These disorders were most pronounced in the 3rd subgroup of stepwise subtotal cerebral ischemia with the shortest interval between dressings, which was 1 day, and in the group of total cerebral ischemia. Conclusion. Thus, the most pronounced decrease in the content of ATP synthase was observed in the groups of total cerebral ischemia, subtotal cerebral ischemia and in the 3rd subgroup of stepwise subtotal cerebral ischemia, with a minimal time interval between the ligation of the common carotid artery. In stepwise subtotal cerebral ischemia with an interval between ligation of the common carotid artery of 7 days, the suppression of the ATP synthase content was not so significant.

ATP synthase in rat brain neurons

Mitochondrial ATP synthase is the main enzyme of ATP synthesis. A large number of diseases, including neurodegenerative ones, are associated with disorders of its assembly and functioning. The aim of the work is to reveal the patterns and features of the regional distribution of ATP synthase in the neurons of the rat brain structures, with a quantitative assessment of its content at the cellular level. The study was carried out on material from 5 outbred white male rats. Paraffin sections were stained by the Nissl method to identify structures of the brain and cervical segments of the spinal cord, and for ATP synthase by immunohistochemical method; 102 structures of the rat brain were studied cytophotometrically. Immunoreactivity of ATP synthase was found in all structures of the rat brain with high heterogeneity, while most structures are characterised with a moderate content of ATP synthase. A high content of ATP synthase was found in large neurons and in neurons of structures of phylogenetically older brain divisions. The content of ATP synthase also depends on the layer of the cortex and the position of neurons in the reflex arc. In the spinal cord the content of ATP synthase depends not only on the size of neurons, but also on the gray matter layer.

Biochemical thresholds for pathological presentation of ATP synthase deficiencies

Mitochondrial ATP synthase is responsible for production of the majority of cellular ATP. Disorders of ATP synthase in humans can be caused by numerous mutations in both structural subunits and specific assembly factors. They are associated with variable pathogenicity and clinical phenotypes ranging from mild to the most severe mitochondrial diseases. To shed light on primary/pivotal functional consequences of ATP synthase deficiency, we explored human HEK 293 cells with a varying content of fully assembled ATP synthase, selectively downregulated to 15–80% of controls by the knockdown of F1 subunits γ, δ and ε. Examination of cellular respiration and glycolytic flux revealed that enhanced glycolysis compensates for insufficient mitochondrial ATP production while reduced dissipation of mitochondrial membrane potential leads to elevated ROS production. Both insufficient energy provision and increased oxidative stress contribute to the resulting pathological phenotype. The threshold for manifestation of the ATP synthase defect and subsequent metabolic remodelling equals to 10–30% of residual ATP synthase activity. The metabolic adaptations are not able to sustain proliferation in a galactose medium, although sufficient under glucose-rich conditions. As metabolic alterations occur when the content of ATP synthase drops below 30%, some milder ATP synthase defects may not necessarily manifest with a mitochondrial disease phenotype, as long as the threshold level is not exceeded.

Role of the mitochondrial ATP synthase central stalk subunits γ and δ in the activity and assembly of the mammalian enzyme

The central stalk of mitochondrial ATP synthase consists of subunits γ, δ, and ε, and along with the membraneous subunit c oligomer constitutes the rotor domain of the enzyme. Our previous studies showed that mutation or deficiency of ε subunit markedly decreased the content of ATP synthase, which was otherwise functionaly and structuraly normal. Interestingly, it led to accumulation of subunit c aggregates, suggesting the role of the ε subunit in assembly of individual enzyme domains. In the present study we focused on the role of subunits γ and δ. Using shRNA knockdown in human HEK293 cells, the protein levels of γ and δ were decreased to 30% and 10% of control levels, respectively. The content of the assembled ATP synthase decreased in accordance with the levels of the silenced subunits, which was also the case for most structural subunits. In contrast, the hydrophobic c subunit was increased to 130% or 180%, respectively and most of it was detected as aggregates of 150–400 kDa by 2D PAGE. In addition the IF1 protein was upregulated to 195% and 300% of control levels. Both γ and δ subunits silenced cells displayed decreased ATP synthase function - lowered rate of ADP-stimulated respiration, a two-fold increased sensitivity of respiration to inhibitor oligomycin, and impaired utilization of mitochondrial membrane potential for ADP phosphorylation. In summary, similar phenotype of γ, δ and ε subunit deficiencies suggest uniform requirement for assembled central stalk as driver of the c-oligomer attachment in the assembly process of mammalian ATP synthase.

OP1 - Molecular mechanisms of mitochondrial protection against oxidative damage in hibernators - the anti-aging effects of heterothermy

Natural hypothermia, in addition to allowing energy saving in hostile conditions, has been associated with delayed aging and increased longevity. However, the molecular basis responsible for observed correlations between the use of daily torpor/hibernation and indices of rate of aging is hitherto unclear. Considering central role of mitochondria dysfunction in the ageing process, we examined several mechanisms that might be involved in mitochondrial protection against oxidative damage during euthermia-hypothermia (and vice versa) transition, in brown adipose tissue (BAT) of the European Ground Squirrel ( Spermophilus citellus ). Results showed that in hibernation increased protein expression of Mn superoxide dismutase coincides with decreased content of ATP synthase and uncoupling protein 1. This suggests that BAT mitochondria during hibernation are protected from oxidative injuries by suppressed oxidative capacity, as well as by upregulated antioxidant defense. Also, the data indicate that such molecular pattern of changes is initiated already during prehibernating period. Namely, in this period we observed accumulations of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor (erythroid 2-related)-like 2, which are probably responsible for suppressed oxidative metabolism, i.e. increased antioxidant capacity, respectively. Increased expression of the mitofusin 1 and detection of the megamitochondria in the prehibernating period indicate intensive mitofusion process in the BAT. This may be another mechanism of protection of mitochondrial content/function during euthermia-hypothermia transition. The results of the study suggest mechanisms that might be associated with increased resistance of “hibernating” mitochondria to the oxidative damage. Also, the data showed that biochemistry responsible for redox balance within the cell involves integration of antioxidant response and transcription control of overall metabolism. Finally, the results go in favor of the previous reports that suggested HIF-1 as a negative modulator of aging.

Alteration of structure and function of ATP synthase and cytochrome c oxidase by lack of Fo-a and Cox3 subunits caused by mitochondrial DNA 9205delTA mutation.

Mutations in the MT-ATP6 gene are frequent causes of severe mitochondrial disorders. Typically, these are missense mutations, but another typeis represented by the 9205delTA microdeletion, which removes the stop codon of the MT-ATP6 gene and affects the cleavage site in the MT-ATP8/MT-ATP6/MT-CO3 polycistronic transcript. This interferes with the processing of mRNAs for the Atp6 (Fo-a) subunit of ATP synthase and the Cox3 subunit of cytochrome c oxidase (COX). Two cases described so far presented with strikingly different clinical phenotypes-mild transient lactic acidosis or fatal encephalopathy. To gain more insight into the pathogenic mechanism, we prepared 9205delTA cybrids with mutation load ranging between 52 and 99% and investigated changes in the structure and function of ATP synthase and the COX. We found that 9205delTA mutation strongly reduces the levels of both Fo-a and Cox3 proteins. Lack of Fo-a alters the structure but not the content of ATP synthase, which assembles into a labile, ∼60kDa smaller, complex retaining ATP hydrolytic activity but which is unable to synthesize ATP. In contrast, lack of Cox3 limits the biosynthesis of COX but does not alter the structure of the enzyme. Consequently, the diminished mitochondrial content of COX and non-functional ATP synthase prevent most mitochondrial ATP production. The biochemical effects caused by the 9205delTA microdeletion displayed a pronounced threshold effect above ∼90% mutation heteroplasmy. We observed a linear relationship between the decrease in subunit Fo-a or Cox3 content and the functional presentation of the defect. Therefore we conclude that the threshold effect originated from a gene-protein level.

HIF-1a and target genes as metabolic biomarkers in Mexican women with breast cancer.

e21063 Background: Breast cancer is the principal malignancy in Mexican women. Breast neoplasias develop chaotic and fragile neovasculature originating intermittent hypoxia which in turn, leads to an adaptive response coordinated by the transcriptional factor HIF-1a (hypoxia-inducible factor-1a). In the present work the expression of HIF-1a was analyzed in different female breast cancer biopsies in order to determine the relationship between HIF-1a expression and cancer prognosis. To determine the functional role of HIF-1a, proteomic and kinetic analysis of several well-established HIF-1 targets such as the glycolytic proteins glucose transporters (GLUT1), hexokinase (HKII), and lactate dehydrogenase (LDH-A) were also determined. In addition, to evaluate the role of HIF-1a expression on mitochondrial function, content of mitochondrial enzymes were also assayed. Methods: Twenty tru-cut biopsies were obtained from female patients (30-45 years old) without previous clinical treatment. After patho-histological assessment of breast cancer incidence, samples were processed by Western-blot analysis (for proteomic analysis) and for enzyme activity determination. Results: Significant increased content of HIF-1a was determined in all human solid tumor biopsies analyzed. In consequence, enhanced GLUT1, HKII and LDH-A contents and activities (12 and 314 mU/mg protein for HK and LDH, respectively) were also detected. On the contrary, mitochondrial enzymes (cytochrome c oxidase, ATP/ADP translocator, glutaminase-K and 2-oxoglutarate dehydrogenase) were not detected, whereas ATP synthase content significantly increased in all analyzed samples. Conclusions: These results clearly indicated that HIF-1a, together with their target glycolytic genes and possibly the mitochondrial enzyme ATP synthase, may be useful to depict a breast cancer metabolic marker pattern.

The Roles of ATP Synthase and the Cytochrome b 6/f Complexes in Limiting Chloroplast Electron Transport and Determining Photosynthetic Capacity

In C(3) plants, CO(2) assimilation is limited by ribulose 1,5-bisphosphate (RuBP) regeneration rate at high CO(2). RuBP regeneration rate in turn is determined by either the chloroplast electron transport capacity to generate NADPH and ATP or the activity of Calvin cycle enzymes involved in regeneration of RuBP. Here, transgenic tobacco (Nicotiana tabacum 'W38') expressing an antisense gene directed at the transcript of either the Rieske iron-sulfur protein of the cytochrome (Cyt) b(6)/f complex or the δ-subunit of chloroplast ATP synthase have been used to investigate the effect of a reduction of these complexes on chloroplast electron transport rate (ETR). Reductions in δ-subunit of ATP synthase content did not alter chlorophyll, Cyt b(6)/f complex, or Rubisco content, but reduced ETR estimated either from measurements of chlorophyll fluorescence or CO(2) assimilation rates at high CO(2). Plants with low ATP synthase content exhibited higher nonphotochemical quenching and achieved higher ETR per ATP synthase than the wild type. The proportional increase in ETR per ATP synthase complex was greatest at 35°C, showing that the ATP synthase activity can vary in vivo. In comparison, there was no difference in the ETR per Cyt b(6)/f complex in plants with reduced Cyt b(6)/f content and the wild type. The ETR decreased more drastically with reductions in Cyt b(6)/f complex than ATP synthase content. This suggests that chloroplast ETR is more limited by Cyt b(6)/f than ATP synthase content and is a potential target for enhancing photosynthetic capacity in crops.

Knockdown of F1 epsilon subunit decreases mitochondrial content of ATP synthase and leads to accumulation of subunit c

The subunit ε of mitochondrial ATP synthase is the only F1 subunit without a homolog in bacteria and chloroplasts and represents the least characterized F1 subunit of the mammalian enzyme. Silencing of the ATP5E gene in HEK293 cells resulted in downregulation of the activity and content of the mitochondrial ATP synthase complex and of ADP-stimulated respiration to approximately 40% of the control. The decreased content of the ε subunit was paralleled by a decrease in the F1 subunits α and β and in the Fo subunits a and d while the content of the subunit c was not affected. The subunit c was present in the full-size ATP synthase complex and in subcomplexes of 200–400kDa that neither contained the F1 subunits, nor the Fo subunits. The results indicate that the ε subunit is essential for the assembly of F1 and plays an important role in the incorporation of the hydrophobic subunit c into the F1-c oligomer rotor of the mitochondrial ATP synthase complex.

The long‐term responses of the photosynthetic proton circuit to drought

Proton motive force (pmf) across thylakoid membranes is not only for harnessing solar energy for photosynthetic CO(2) fixation, but also for triggering feedback regulation of photosystem II antenna. The mechanisms for balancing these two roles of the proton circuit under the long-term environmental stress, such as prolonged drought, have been poorly understood. In this study, we report on the response of wild watermelon thylakoid 'proton circuit' to drought stress using both in vivo spectroscopy and molecular analyses of the representative photosynthetic components. Although drought stress led to enhanced proton flux via a approximately 34% increase in cyclic electron flow around photosystem I (PS I), an observed approximately fivefold decrease in proton conductivity, g(H)(+), across thylakoid membranes suggested that decreased ATP synthase activity was the major factor for sustaining elevated q(E). Western blotting analyses revealed that ATP synthase content decreased significantly, suggesting that quantitative control of the complex plays a pivotal role in down-regulation of g(H)(+). The expression level of cytochrome b(6)f complex - another key control point in photosynthesis - also declined, probably to prevent excess-reduction of PS I electron acceptors. We conclude that plant acclimation to long-term environmental stress involves global changes in the photosynthetic proton circuit, in which ATP synthase represents the key control point for regulating the relationship between electron transfer and pmf.

TMEM70 protein — A novel ancillary factor of mammalian ATP synthase

An increasing number of patients with nuclear genetic defects of mitochondrial ATP synthase have been identified in recent years. They are characterized by early onset, lactic acidosis, 3-methylglutaconic aciduria, hypertrophic cardiomyopathy and encephalopathy and most cases have a fatal outcome. Patient tissues show isolated defect of the ATP synthase complex and its content decreases to ≥30% of normal due to altered enzyme biosynthesis and assembly. Gene mapping and complementation studies have identified mutations in TMEM70 gene encoding a 30kD mitochondrial protein of unknown function as the cause of the disease. An altered synthesis of this new ancillary factor in ATP synthase biogenesis was found in most of the known patients with decreased ATP synthase content. As revealed by phylogenetic analysis, TMEM70 is specific for higher eukaryotes.

Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency.

BackgroundTo strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes.ResultsMolecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for nuclear defects.ConclusionOur analysis showed that deficiency in the ATP synthase protein complex amount is generally accompanied by only minor changes in expression of ATP synthase related genes. It also suggested that the site (mtDNA vs nuclear DNA) and the severity (ATP synthase content) of the underlying defect have diverse effects on cellular gene expression phenotypes, which warrants further investigation of cell cycle regulatory and signal transduction pathways in other OXPHOS disorders and related pharmacological models.

Adenine nucleotide translocator promotes oxidative phosphorylation and mild uncoupling in mitochondria after dexamethasone treatment

The composition of the mitochondrial inner membrane and uncoupling protein [such as adenine nucleotide translocator (ANT)] contents are the main factors involved in the energy-wasting proton leak. This leak is increased by glucocorticoid treatment under nonphosphorylating conditions. The aim of this study was to investigate mechanisms involved in glucocorticoid-induced proton leak and to evaluate the consequences in more physiological conditions (between states 4 and 3). Isolated liver mitochondria, obtained from dexamethasone-treated rats (1.5 mg.kg(-1).day(-1)), were studied by polarography, Western blotting, and high-performance thin-layer chromatography. We confirmed that dexamethasone treatment in rats induces a proton leak in state 4 that is associated with an increased ANT content, although without any change in membrane surface or lipid composition. Between states 4 and 3, dexamethasone stimulates ATP synthesis by increasing both the mitochondrial ANT and F1-F0 ATP synthase content. In conclusion, dexamethasone increases mitochondrial capacity to generate ATP by modifying ANT and ATP synthase. The side effect is an increased leak in nonphosphorylating conditions.

Modulation of Charge in the Phosphate Binding Site of Escherichia coli ATP Synthase

This paper presents a study of the role of positive charge in the P(i) binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P(i) binding sites of proteins generally and in the P(i) binding site of the betaE catalytic site of ATP synthase specifically. Removal of natural betaArg-246 (betaR246A mutant) abrogates P(i) binding; restoration of P(i) binding was achieved by mutagenesis of either residue betaAsn-243 or alphaPhe-291 to Arg. Both residues are located in the P(i) binding site close to betaArg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of betaArg-246 retained P(i) binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of betaArg-246 in betaR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in betaN243R/betaR246A and alphaF291R/betaR246A. BetaArg-243 or alphaArg-291 in presence of betaArg-246 was less effective; the combination of alphaF291R/betaN243R with natural betaArg-246 was just as detrimental as betaR246A. The data demonstrate that electrostatic interaction is an important component of initial P(i) binding in catalytic site betaE and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.

Mitochondrial uncoupling proteins – facts and fantasies

Instead of a comprehensive review, we describe the basic undisputed facts and a modest contribution of our group to the fascinating area of the research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, protein-mediated uncoupling, we discuss the assumption that due to their low abundance the novel mitochondrial uncoupling proteins (UCP2 to UCP5) can provide only a mild uncoupling, i.e. can decrease the proton motive force by several mV only. Contrary to this, the highly thermogenic role of UCP1 in brown adipose tissue is not given only by its high content (approximately 5 % of mitochondrial proteins) but also by the low ATP synthase content and high capacity respiratory chain. Fatty acid cycling mechanism as a plausible explanation for the protonophoretic function of all UCPs and some other mitochondrial carriers is described together with the experiments supporting it. The phylogenesis of all UCPs, estimated UCP2 content in several tissues, and details of UCP2 activation are described on the basis of our experiments. Functional activation of UCP2 is proposed to decrease reactive oxygen species (ROS) production. Moreover, reaction products of lipoperoxidation such as cleaved hydroperoxy-fatty acids and hydroxy-fatty acid can activate UCP2 and promote feedback down-regulation of mitochondrial ROS production.

Myocardial force development and structural changes associated with monocrotaline induced cardiac hypertrophy and heart failure.

In this study alterations are characterized which occur, in myocardial force development morphological appearance and protein composition, during the development of cardiac hypertrophy and heart failure in monocrotaline (MCT) treated rats. The transition from cardiac hypertrophy to heart failure was studied by comparing the results from control (CON) and two MCT groups (40 and 44 mg/kg body weight). The three experimental groups consisted of at least five animals each. Parameters studied were: body weight (measured daily), lung/body weight ratio, right ventricular wall volume and thickness, and force development in thin right ventricular trabeculae at 27 degrees C, using different extracellular calcium concentrations and pacing frequencies. MCT injection resulted in marked right ventricular hypertrophy and heart failure as evidenced by an up to 2-fold increase in lung/body weight ratio and a 1.7-fold increase in wall volume. The MCT groups showed a negative force-frequency relation and maximum force was up to 2-fold less than in the CON group. Protein analysis by means of one- and two-dimensional gel electrophoresis revealed a marked (7-fold) up-regulation of the slow myosin heavy chain isoform as well as a 4.5-fold increase in the content of the cytoskeletal protein desmin, whereas the mitochondrial protein ATP-synthase content was reduced. Hence MCT-induced cardiac hypertrophy and heart failure result in altered cellular calcium handling, depression of maximum force output, an increase in the economy of myocardial contraction and changes in cytoskeletal structure and energy supply.