Mitochondrial ATP Synthase Activity Research Articles

Energetics of the reaction of DDT with the ATP synthase of inesct mitochondria

In a previous paper (Hassan M Younis et al. Biotechnol Appl Biochem. “2002” 35: 9- 17)we have shown that the insecticide DDT exerts its action through its inhibition of the mitochondrial ATP synthase activity. Here I report on the kinetics and equilirium constants of the reaction between DDT and the purified enzyme in vitro. Analysis of the temperature dependence of kinetics and equilibrium constants allowed determination of the energetics of binding of DDT to the enzyme. The dissociation constant (Kd) for DDT bound to the enzyme was determined, and the changes in free energy, enthalpy and entropy that are associated with binding of DDT to The enzyme were all calculated. the significance of the results will be discussed in terms of a knowledge -based approach for development of better insecticides.

Mitochondrial Encephalomyopathy inDrosophila

Mitochondrial encephalomyopathies are common and devastating multisystem genetic disorders characterized by neuromuscular dysfunction and tissue degeneration. Point mutations in the human mitochondrial ATP6 gene are known to cause several related mitochondrial disorders: NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh's syndrome), and FBSN (familial bilateral striatal necrosis). We identified a pathogenic mutation in the Drosophila mitochondrial ATP6 gene that causes progressive, adult-onset neuromuscular dysfunction and myodegeneration. Our results demonstrate ultrastructural defects in the mitochondrial innermembrane, neural dysfunction, and a marked reduction in mitochondrial ATP synthase activity associated with this mutation. This Drosophila mutant recapitulates key features of the human neuromuscular disorders enabling detailed in vivo studies of these enigmatic diseases.

The effects of simulated microgravity on cultured chicken embryonic chondrocytes

Using the cultured chicken embryonic chondrocytes as a model, the effects of simulated microgravity on the microtubular system of the cellular skeleton, extracellular matrix, alkaline phosphatase activity, intracellular free calcium concentration and mitochondrial ATP synthase activity with its oligomycin inhibition rate were studied with a clinostat. The microtubular content was measured by a flow cytometer. The decrease of microtubular content showed the impairment of the cellular skeleton system. Observation on the extracellualr matrix by the scanning electron microscopy showed that it decreased significantly after rotating, and the fibers in the extracellular matrix were more tiny and disorderly than that of the control group. It can be concluded that the simulated microgravity can affect the secreting and assembly of the extracellular matrix. In contrast to the control, there was a time course decrease in alkaline phosphatase activity of chondrocytes, a marker of matrix mineralization. Meanwhile a significant drop in the intracellular calcium concentration happened at the beginning of rotation. These results indicate that simulated microgravity can suppress matrix calcification of cultured chondrocytes, and intracellular free calcium may be involved in the regulation of matrix calcification as the second signal transmitter. No significant changes happened in the mitochondrial ATP synthase activity and its oligomycin inhibition rate. Perhaps the energy metabolism wasn't affected by the simulated microgravity. The possible mechanisms about them were discussed.

Regulation of the mitochondrial ATP-synthase in health and disease

In most tissues the mitochondrial ATP-synthase plays a central role by synthesizing the bulk of ATP. According to the classical theory of respiratory control, flux through this enzyme is solely determined by substrate (ADP) concentration while the enzyme has a fixed capacity. However, in different cell types such as rat cardiomyocytes and neurons, dog heart, human fibroblasts, and skeletal and heart muscle from children, it has been shown that active regulation of the mitochondrial ATP-synthase in response to cellular energy demand exists. For example, in rat cardiomyocytes the mitochondrial ATP-synthase activity is down-regulated in response to anoxia or mitochondrial uncoupling. By this mechanism cellular ATP is conserved, as under these conditions the ATP-synthase would work in reverse and hydrolyze ATP. When cardiomyocytes are stimulated to contract, ATP-synthase activity is up-regulated in line with the increased energy demand. Preincubation of the cardiomyocytes with positive inotropic substances results in further up-regulation of the ATP-synthase. By blocking calcium transport, it has been shown that the up-regulation of the enzyme is calcium-dependent. On a molecular level, up-regulation is probably mediated by the calcium-binding inhibitor protein (CaBI) and down-regulation via the inhibitor protein IF 1. The ATP-synthase system is disturbed under several pathophysiological conditions. First, mutations can cause a primary defect in the mitochondrial ATP-synthase (respiratory chain defect). Furthermore, secondary defects of the ATP-synthase occur. In rat models abnormalities of ATP-synthase can be detected in different types of cardiomyopathy/heart hypertrophy. The changes are reversible in response to treatment of the heart diseases. Abnormalities of the ATP-synthase system can be observed in fibroblasts from patients with neuronal ceroidlipofuscinoses. Toxic metabolites accumulating in methylmalonic acidurias can inhibit ATP-synthase. When neurons are incubated with 3-OH glutarate — a substance accumulating in glutaric aciduria I—as a model for glutaric aciduria I, ATP-synthase activity is compromised. This lack of energy may lead to ‘slow onset’ excitotoxicity and finally cell death. Cells can be rescued by adding creatine to the incubation medium. In d-2-hydroxyglutaric aciduria, inhibition of the ATP-synthase has been observed.

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

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

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

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

Overproduction of PDR3 suppresses mitochondrial import defects associated with a TOM70 null mutation by increasing the expression of TOM72 in Saccharomyces cerevisiae.

Most mitochondrial proteins are synthesized with cleavable amino-terminal targeting signals that interact with the mitochondrial import machinery to facilitate their import from the cytosol. We previously reported that the presequence of the F(1)-ATPase beta subunit precursor (pre-F(1)beta) acts as an intramolecular chaperone that maintains the precursor in an import-competent conformation prior to import (P. Hajek, J. Y. Koh, L. Jones, and D. M. Bedwell, Mol. Cell. Biol. 17:7169-7177, 1997). We also found that a mutant form of pre-F(1)beta with a minimal targeting signal (Delta 1,2 pre-F(1)beta) is inefficiently imported into mitochondria because it rapidly folds into an import-incompetent conformation. We have now analyzed the consequences of reducing the pre-F(1)beta targeting signal to a minimal unit in more detail. We found that Delta 1,2 pre-F(1)beta is more dependent upon the Tom70p receptor for import than WT pre-F(1)beta is, resulting in a growth defect on a nonfermentable carbon source at 15 degrees C. Experiments using an in vitro mitochondrial protein import system suggest that Tom70p functions to maintain a precursor containing the Delta 1,2 pre-F(1)beta import signal in an import-competent conformation. We also identified PDR3, a transcriptional regulator of the pleiotropic drug resistance network, as a multicopy suppressor of the mitochondrial import defects associated with Delta 1,2 pre-F(1)beta in a tom70 Delta strain. The overproduction of PDR3 mediated this effect by increasing the import of Delta 1,2 pre-F(1)beta into mitochondria. This increased the mitochondrial ATP synthase activity to the extent that growth of the mutant strain was restored under the selective conditions. Analysis of the transcription patterns of components of the mitochondrial outer membrane import machinery demonstrated that PDR3 overproduction increased the expression of TOM72, a little studied TOM70 homologue. These results suggest that Tom72p possesses overlapping functions with Tom70p and that the pleiotropic drug resistance network plays a previously unappreciated role in mitochondrial biogenesis.

Severe Energy Impairment Consequent to Inactivation of Mitochondrial ATP Synthase as an Early Event in Cell Death: A Mechanism for the Selective Sensitivity to H 2O 2 of Differentiating Erythroleukemia Cells

Irreversible damage to Friend’s erythroleukemia cells was caused by induction of endogenous heme biosynthesis with the differentiating agent N,N′-hexamethylene bisacetamide followed by a 30-min exposure to 0.25 mM H 2O 2. Early irreversible ATP depletion was observed concomitant with oxidative inactivation of the mitochondrial ATP synthase. Cell proliferative capacity was also impaired within 2 h of the treatment, and progressive delayed cell lethality, starting 2 h after the insults, was also found. Based on the prevention provided by specific antioxidants and on the absence of malodialdehyde production, all the effects were ascribed to the oxidant action of ·OH radicals, or closely related species, generated through iron-catalyzed reactions of H 2O 2, which apparently caused site-directed oxidative modifications of iron-binding proteins, in particular mitochondrial ATP synthase, rather than peroxidation of membrane lipids. Similar effects were mimicked even in the parental cell line when oligomycin was used to inhibit selectively mitochondrial ATP synthase activity, thereby lowering the enzyme activity to a level similar to that found in H 2O 2-damaged differentiating cells. Hence, induction of erythroid differentiation makes the mitochondrial ATP synthase a major target of H 2O 2 by enhancing the availability of redox-active iron in the local environment of the enzyme. Subsequent oxidative inactivation of the mitochondrial ATP synthase, resulting in severe energy impairment, leads to loss of cell growth capacity. Erythroleukemia cells may serve as a model system for the combination of two selective properties: (1) the capacity for carrying out efficient heme synthesis and/or for undergoing iron overload-like state; and (2) subsequent enhanced sensitivity to reactive oxygen species generators. Early severe mitochondrial dysfunction and energy impairment may be a major part of the mechanism of the sensitivity.

Mitochondrial dysfunction after fetal alcohol exposure.

Specific mitochondrial enzyme activities and mRNA levels were measured in the heart, brain, and liver tissues of a group of 1-day-old neonatal rats whose mothers were alcohol-fed during pregnancy and compared with a control group. The results show a significant decrease in mitochondrial ATP synthase activity in both the brain and liver, as well as a decrease in complex III activity in the liver of rats exposed to alcohol. Other mitochondrial enzymes activities (e.g., citrate synthase, cytochrome c oxidase, and complex I), as well as specific mitochondrial transcript levels, were not significantly affected. Heart mitochondrial enzyme activities were not significantly affected. These data reveal that a tissue-specific response occurs after fetal exposure to alcohol and may explain some of the cellular events occurring in fetal alcohol syndrome resulting in abnormal growth and neurological development.

Regulation of the mitochondrial ATP synthase is defective in rat heart during alcohol-induced cardiomyopathy

Mitochondrial AtP synthase capacity was measured in cardiomyocytes from sucrose-fed (control) and alcohol-fed (cardiomyopathic) rats. Cells from alcohol-fed rats showed an ATP synthase capacity raised by 40% relative to the control values of 1.8 μmol/min per mg cell protein. Cells from control rats were able to increase their ATP synthase capacity by up to 80% in response to increased energy demand. In contrast, cells from alcohol-fed rats had lost the ability to up-regulate their ATP synthase. Cells from control rats maintained their internal ATP levels at 38 nmol/mg cell protein before and after an increased energy demand. In cells from alcohol-fed rats, ATP levels fell by 20 after 2 min of increased energy demand. It was concluded that the inability of cells from alcohol-fed rats to maintain ATP levels was due to their reduced ability to increased mitochondrial ATP synthase activity as energy demand is increased. Such ATP deficits may contribute to heart dysfunction in alcohol-induced cardiomyopathy.

Mitochondrial ATP-synthase activity in cardiomyocytes after aerobic-anaerobic metabolic transition.

It has been hypothesized that, in oxygen-depleted myocardial cells, mitochondria are depolarized and the F1,F0-proton adenosinetriphosphatase (ATPase) catalyzes net ATP hydrolysis when the cells exhibit the signs of an aerobic-anaerobic metabolic transition, which are increased lactate formation and decline in high-energy phosphate reserves [W. Rouslin, C. W. Broge, and I. L. Grupp. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1759-H1766, 1990]. This hypothesis was tested by incubating isolated cardiomyocytes from the adult rat in substrate-free Tyrode solution (37 degrees C, pH 7.4) at a PO2 less than or equal to 0.1 Torr, i.e., 1,000-fold below the normal arterial level. At this deep hypoxia, the following results were found. 1) Lactate production was activated to maximal rates and high-energy phosphate contents decreased (aerobic-anaerobic metabolic transition). The inhibitor of the mitochondrial F1,F0-proton ATPase oligomycin, however, added upon establishment of hypoxia, did not slow down, as in the case of depolarized mitochondria, but moderately accelerated energy depletion. 2) Activation of mitochondrial ATP hydrolysis could be provoked in these hypoxic cells by addition of cyanide, antimycin A, and rotenone, i.e., specific inhibitors of certain sites of the respiratory chain. The enhancement of loss of ATP could be inhibited by oligomycin. The results demonstrate that states of deep hypoxia of the cardiomyocyte are possible in which it undergoes an aerobic-anaerobic metabolic transition, indicated by increased lactate formation and progressive loss of cellular energy reserves, and yet mitochondrial ATPase hydrolytic activity is not activated.

Mitochondrial ATP-synthase activity in cardiomyocytes after aerobic-anaerobic metabolic transition: H. Michael Piper, Axel Koop, Thomas Noll. Institute of Physiology, University of Düsseldorf, Moorenstr. 5, D-4000 Düsseldorf, Germany

Mitochondrial ATP-synthase activity in cardiomyocytes after aerobic-anaerobic metabolic transition: H. Michael Piper, Axel Koop, Thomas Noll. Institute of Physiology, University of Düsseldorf, Moorenstr. 5, D-4000 Düsseldorf, Germany