Mitochondrial Bound Hexokinase Activity as a Preventive Antioxidant Defense (original) (raw)
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Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (m) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexoki-nase activity also regulated m , because glucose stabilized low m values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the m and in H 2 O 2 generation. The glucose analogue 2-de-oxyglucose completely impaired H 2 O 2 formation in state 3-state 4 transition. In sharp contrast, the mt-hexoki-nase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochon-dria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H 2 O 2 generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellu-lar glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.
The functional compartmentation of mitochondrial hexokinase
Archives of Biochemistry and Biophysics, 1974
These studies examined the functiona. relationship between rat hepatic mitochondria and associated hexokinase (ATP: o-hexose&phosphotransferase, 2.7.1.1) to determine whether the binding of hexokinase to mitochondria might provide a privileged interaction with sites of ATP production. Initial kinetic analysis followed the sequential flow of phosphate through ATP generated by the mitochondria into glucose-6-phosphate catalyzed by the bound hexokinase. Kinetics were compared with an identical bound hexokinase-mitochon drial system using externally supplied ATP. The hexokinase had lower apparent K, values for ATP generated in the mitochondria from supplied ADP than for ATP provided. Respiratory inhibitors blocked both the ADP-and ATP-mediated reactions. Tracer studies further documented that the mitochondrial hexokinase initially and preferentially utilized the internally generat.ed nucleotide. These studies demonstrate that the act,ive site of bound hexokinase is relatively inaccessible to extramitochondrial ATP. They provide evidence that bound hexokinase can sequentially accept mitochondrially generated ATP in a kinetically advantageous way. Finally, they support the assumption that mitochondrial binding of this acceptor enzyme may play a propitious role in cellular energy economy.
Mitochondrial Reactive Oxygen Species and the
2014
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Mitochondrial function is differentially affected upon oxidative stress
Free Radical Biology and Medicine, 1999
The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in synaptosomes treated with ascorbate/iron. A loss of membrane integrity, evaluated by electron microscopy and by LDH leakage, was observed in peroxidized synaptosomes and it was prevented by pre-incubation with vitamin E (150 M) and idebenone (50 M). ATP levels decreased, in synaptosomes exposed to ascorbate/iron, as compared to controls. NADH-ubiquinone oxidoreductase (Cx I) and cytochrome c oxidase (Cx IV) activities were unchanged after ascorbate/ iron treatment, whereas succinate-ubiquinone oxidoreductase (Cx II), ubiquinol cytochrome c reductase (Cx III) and ATP-synthase (Cx V) activities were reduced by 55%, 40%, and 55%, respectively. The decrease of complex II and ATP-synthase activities was prevented by reduced glutathione (GSH), whereas the other antioxidants tested (vitamin E and idebenone) were ineffective. However, vitamin E, idebenone and GSH prevented the reduction of complex III activity observed in synaptosomes treated with ascorbate/iron. GSH protective effect suggests that the oxidation of protein SH-groups is involved in the inhibition of complexes II, III and V activity, whereas vitamin E and idebenone protection suggests that membrane lipid peroxidation is also involved in the reduction of complex III activity. These results may indicate that the inhibition of the mitochondrial respiratory chain enzymatic complexes, that are differentially affected by oxidative stress, can be recovered by specific antioxidants.
Inhibition of NADH-Linked Mitochondrial Respiration by 4-Hydroxy-2-nonenal †
Biochemistry, 1998
During the progression of certain degenerative conditions, including myocardial ischemiareperfusion injury, mitochondria are a source of increased free-radical generation and exhibit declines in respiratory function(s). It has therefore been suggested that oxidative damage to mitochondrial components plays a critical role in the pathology of these processes. Polyunsaturated fatty acids of membrane lipids are prime molecular targets of free-radical damage. A major product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is highly cytotoxic and can readily react with and damage protein. In this study, the effects of HNE on intact cardiac mitochondria were investigated to gain insight into potential mechanisms by which free radicals mediate mitochondrial dysfunction. Exposure of mitochondria to micromolar concentrations of HNE caused rapid declines in NADH-linked but not succinate-linked state 3 and uncoupled respiration. The activity of complex I was unaffected by HNE under the conditions of our experiments. Loss of respiratory activity reflected the inability of HNE-treated mitochondria to meet NADH demand during maximum rates of O 2 consumption. HNE exerted its effects on intact mitochondria by inactivating R-ketoglutarate dehydrogenase. These results therefore identify a potentially important mechanism by which free radicals bring about declines in mitochondrial respiration. Abstract published in AdVance ACS Abstracts, December 15, 1997.
An Approach Based on Mitochondrial-Targeted Antioxidants
2015
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Redox Control of Mitochondrial Functions
Antioxidants & Redox Signaling, 2001
Redox reactions and electron flow through the respiratory chain are the hallmarks of mitochondria. By supporting oxidative phosphorylation and metabolite transport, mitochondrial redox reactions are of central importance for cellular energy conversion. In the present review, we will discuss two other aspects of the mitochondrial redox state: (i) its control of mitochondrial Ca 21 homeostasis, and (ii) the intramitochondrial formation of reactive oxygen or nitrogen species that strongly influence electron flow of the respiratory chain. Antioxid. Redox Signal. 3, 515-523. 515 REDOX REGULATION IN MITOCHONDRIA: THE CASE OF Ca 21 HOMEOSTASIS Reactive oxygen species formation and the redox barrier in mitochondria 2 production in state 4 compared with the state 3 of respiration.
Regulation and Cytoprotective Role of Hexokinase III
PLoS ONE, 2010
Background: Hexokinases (HKs) catalyze the first step in glucose metabolism. Of the three mammalian 100-kDa HK isoforms, HKI and II can bind to mitochondria and protect against cell death. HKIII does not bind mitochondria, and little is known about its regulation or cytoprotective effects. We studied the regulation of HKIII at the transcriptional and protein levels and investigated its role in cellular protection. Methodology/Principal Findings: We show that like HKII, HKIII expression is regulated by hypoxia, but other factors that regulate HKII expression have no effect on HKIII levels. This transcriptional regulation is partially dependent on hypoxiainducible factor (HIF) signaling. We also demonstrate regulation at the protein level, as mutations in putative N-terminal substrate binding residues altered C-terminal catalytic activity, suggesting that HKIII activity is governed, in part, by interactions between these two domains. Overexpression of HKIII reduced oxidant-induced cell death, increased ATP levels, decreased the production of reactive oxygen species (ROS), and preserved mitochondrial membrane potential. HKIII overexpression was also associated with higher levels of transcription factors that regulate mitochondrial biogenesis, and greater total mitochondrial DNA content. Attempts to target HKIII to the mitochondria by replacing its N-terminal 32-aminoacid sequence with the mitochondrial-targeting sequence of HKII led to protein aggregation, suggesting that this region is necessary to maintain proper protein folding and solubility. Conclusions/Significance: These results suggest that HKIII is regulated by hypoxia and there are functional interactions between its two halves. Furthermore, HKIII exerts protective effects against oxidative stress, perhaps by increasing ATP levels, reducing oxidant-induced ROS production, preserving mitochondrial membrane potential, and increasing mitochondrial biogenesis.
Bioflavonoid regulation of ATPase and hexokinase activity in ehrlich ascites cell mitochondria
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1977
The mitochondrial ATPase (EC 3.6.1.3) Ehrlich ascites cell mitochondria, was inhibited by D-glucose under physiological concentrations of ATP. The generation of ADP by the mitochondrial bound hexokinase, seems to be the reason for the D-glucose inhibitory effect. Reversal of the inhibitory effect of ADP on Ehrlich ascites cell mitochondria ATPase by an ATP-regenerating system was achieved. (2) Dissociation of mitochondrial bound hexokinase from the mitochondria eliminated the inhibitory effect of D-glucose. Rebinding of the hexokinase to the mitochondria regenerated the D-glucose inhibitory effect on Ehrlich ascites cell mitochondria ATPase. (3) Bioflavonoids such as quercetin inhibit the mitochondrial hexokinase activity, but do not change the mitochondrial ATPase activity of isolated Ehrlich ascites tumor cell mitochondria. (4) The inhibitory effect of bioflavonoids on mitochondrial bound hexokinase activity is shown to be dissociable from the ascites tumor cell mitochondria and seems to be associated with regulatory rather than catalitic sites of the enzyme.