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Revisado por pares
Mitochondrial Bound Hexokinase Activity as a Preventive Antioxidant Defense
2004; Elsevier BV; Volume: 279; Issue: 38 Linguagem: Inglês
10.1074/jbc.m403835200
ISSN1083-351X
AutoresWagner Seixas da‐Silva, Armando Gómez‐Puyou, Marietta Tuena de Gómez‐Puyou, Rafael Moreno‐Sánchez, Fernanda G. De Felice, Leopoldo de Meis, Marcus F. Oliveira, Antônio Galina,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoBrain 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 (Δψm) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Δψm, because glucose stabilized low Δψm 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 Δψm and in H2O2 generation. The glucose analogue 2-deoxyglucose completely impaired H2O2 formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-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 mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H2O2 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 intracellular 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. 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 (Δψm) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Δψm, because glucose stabilized low Δψm 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 Δψm and in H2O2 generation. The glucose analogue 2-deoxyglucose completely impaired H2O2 formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-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 mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H2O2 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 intracellular 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. Glucose is an essential molecule for maintaining life as it is a major metabolic fuel, which, through its degradation via glycolysis and subsequent oxidative phosphorylation, generates high energy phosphate compounds responsible for driving many cellular processes. Normal levels of glucose and growth factors may protect cells from apoptotic events (1Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. 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Although the mechanism of glucose toxicity is not completely understood, many studies have shown that high glucose increases the formation of advanced glycation end products (6Brownlee M. Annu. Rev. Med. 1995; 46: 223-234Crossref PubMed Scopus (1174) Google Scholar), glucose flux through the aldose reductase pathway (7Lee A.Y. Chung S.K. Chung S.S. Proc. Natl. Acad. Sci. U. S. 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It has been proposed that the production of ROS by mitochondria via the respiratory chain is a causal link between high glucose and the main pathways responsible for hyperglycemic damage (3Russell J.W. Golovoy D. Vincent A.M. Mahendru P. Olzmann J.A. Mentzer A. Feldman E.L. FASEB J. 2002; 16: 1738-1748Crossref PubMed Scopus (429) Google Scholar, 8Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3761) Google Scholar). Transport and the first step of glucose utilization within the cells is catalyzed by hexokinase (EC 2.7.1.1), which participates in blood glucose homeostasis. In mammals, there are four isoforms of hexokinase (hexokinase-I to hexokinase-IV), differing in their affinities for glucose and inhibition by glucose 6-phosphate (Glc-6-P) and Pi as well as in their subcellular distribution (9Wilson J.E. J. Bioenerg. 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Hexokinase-I and hexokinase-II bind to mitochondria, and the extent of their association varies from tissue to tissue, with brain, kidney, cardiac, and skeletal muscle and tumor cells being the sites that display a larger percentage of mitochondrial association (15Bustamante E. Pedersen P.L. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 3735-3739Crossref PubMed Scopus (351) Google Scholar, 16Parry D.M. Pedersen P.L. J. Biol. Chem. 1984; 259: 8917-8923Abstract Full Text PDF PubMed Google Scholar, 17Viitanen P.V. Geiger P.J. Erickson-Viitanen S. Bessman S.P. J. Biol. Chem. 1984; 259: 9679-9686Abstract Full Text PDF PubMed Google Scholar, 18Bustamante E. Morris H.P. Pedersen P. J. Biol. Chem. 1981; 256: 8699-8704Abstract Full Text PDF PubMed Google Scholar, 19Schwab D.A. Wilson J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2563-2567Crossref PubMed Scopus (128) Google Scholar). It has been suggested that this binding takes place at the voltage-dependent anion channels (VDACs), also known as mitochondrial porins (20Cesar M.D.C. Wilson J.E. Arch. Biochem. Biophys. 2004; 422: 191-196Crossref PubMed Scopus (58) Google Scholar, 21Nakashima R.A. Mangan P.S. Colombini M. Pedersen P.L. Biochemistry. 1986; 25: 1015-1021Crossref PubMed Scopus (195) Google Scholar). Associated with VDAC is the ADP/ATP carrier (or adenine nucleotide translocator, ANT), which allows the exchange of ADP and ATP through the inner mitochondrial membrane (22Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (347) Google Scholar, 23Vyssokikh M.Y. Brdiczka D. Acta Biochim. Pol. 2003; 50: 389-404Crossref PubMed Scopus (148) Google Scholar). Interestingly, recent studies indicated that mt-hexokinase activity inhibits not only the Bax-induced cytochrome c release and apoptosis in HeLa cells but also early apoptotic events mediated by Akt/protein kinase B activation (1Gottlob K. Majewski N. Kennedy S. Kandel E. Robey R.B. Hay N. Genes Dev. 2001; 15: 1406-1418Crossref PubMed Scopus (789) Google Scholar, 24Pastorino J.G. Shulga N. Hoek J.B. J. Biol. Chem. 2002; 277: 7610-7618Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). It has been very well established that 1-2% of the consumed O2 by the respiratory chain is diverted to generate ROS such as superoxide ( O2−˙ and hydrogen peroxide (H2O2); these side reactions are mainly catalyzed by the respiratory complexes I (25Cadenas E. Boveris A. Ragan C.I. Stopani A.O. Arch. Biochem. Biophys. 1977; 180: 248-257Crossref PubMed Scopus (702) Google Scholar, 26Turrens J.F. Boveris A. Biochem. 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Pharmacol. 1997; 54: 533-539Crossref PubMed Scopus (208) Google Scholar, 39Giasson B.I. Ischiropoulos H. Lee V.M. Trojanowski J.Q. Free Radic. Biol. Med. 2002; 32: 1264-1275Crossref PubMed Scopus (254) Google Scholar). The physiological rate of mitochondrial ROS production is inversely proportional to the availability of cytosolic ADP (30Cadenas E. Davies K.J. Free Radic. Biol. Med. 2000; 29: 222-230Crossref PubMed Scopus (2465) Google Scholar). Thus, a diminution in the ADP levels induces an increase in the magnitude of the mitochondrial membrane potential (Δψm), which, in turn, decreases the respiratory rate, leading to stimulation of ROS generation due to the highly reduced state of the components of the electron transport chain. When the mitochondrial ADP levels rise, the inverse occurs. This has been attributed to the reduction of the Δψm through F1F0 ATP synthase complex activity. In fact, mitochondrial ROS generation is strongly dependent on Δψm levels, because high H+ gradients increase O2−˙ and H2O2 formation (40Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1441) Google Scholar). On the other hand, a small decrease in Δψm levels strongly diminishes H2O2 production (40Korshunov S.S. Skulachev V.P. Starkov A.A. FEBS Lett. 1997; 416: 15-18Crossref PubMed Scopus (1441) Google Scholar). Therefore, in the present work the relationship between Δψm and ROS production with the ADP generated by the mt-hexokinase activity was evaluated in both isolated rat brain mitochondria and rat embryonic cortical neurons. The results suggested that mt-hexokinase activity was directly involved in the local ADP recycling mechanism, providing a novel physiological antioxidant role in rat neuronal cells. Chemicals—ADP, ATP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), scopoletin, horseradish peroxidase, rotenone, safranine O, P1,P5-di(adenosine 5′)-pentaphosphate (Ap5A), 2-deoxyglucose (2-DOG), 6-deoxyglucose (6-DOG), N-acetyl cysteine (NAC), yeast hexokinase, Glc-6-P dehydrogenase (Leuconostoc mesenteroides), Percoll, fatty acid-free bovine serum albumin, NAD+, polyornitine, RPMI 1640 medium, and Glc-6-P were purchased from Sigma-Aldrich. Hydrogen peroxide was from Merck. Neurobasal medium was from Invitrogen. CM-H2DCFDA was from Molecular Probes (Eugene, OR). All other reagents were from analytical grade. Animals and Mitochondrial Isolation—Adult male Wistar rats weighing 200-230 g fasted overnight prior to being killed by decapitation. Mitochondria were isolated by conventional differential centrifugation from forebrains as described elsewhere (41Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (346) Google Scholar) and kept at 4 °C throughout the isolation procedure. Briefly, brains from two rats were rapidly removed to an ice-cold isolation buffer containing 0.32 m sucrose, 1 mm EDTA, 1 mm EGTA, and 10 mm Tris-HCl (pH 7.4). After five washes to remove contaminating blood, the tissue was sliced into little pieces in isolation buffer. The tissue was manually homogenized during two cycles of 10 s in a Teflon glass potter. The homogenate was centrifuged at 1.330 × g for 3 min in a Hitachi Himac SCR20B RPR 20-2 rotor. The supernatant was carefully removed and centrifuged at 21,200 × g for 10 min. The pellet was re-suspended in isolation buffer (10 ml/g tissue originally homogenized) and divided in two equal volumes. To obtain the mt-hexokinase-depleted mitochondria, one fraction of the 21,200 × g pellet was mixed with 2 mm Glc-6-P (42BeltrandelRio H. Wilson J.E. Arch. Biochem. Biophys. 1991; 286: 183-194Crossref PubMed Scopus (54) Google Scholar). The other fraction was used as a control. Both fractions were incubated in an ice bath for 30 min and then centrifuged at 21,200 × g for 10 min. The supernatants were gently removed and stored for hexokinase activity. Alternatively, 2 mm Glc-6-P was included in the isolation buffer from the beginning of the isolation procedure and kept throughout all steps. The pellets obtained were re-suspended in 15% Percoll. The discontinuous density gradient was prepared manually by layering 3-ml fractions of the resuspended pellet on two preformed layers consisting of 3.5 ml of 23% Percoll above 3.5 ml of 40% Percoll. Tubes were centrifuged for 5 min at 30,700 × g with slow brake deceleration. The material equilibrating near the interface between the 23 and 40% Percoll layers was gently diluted 1:4 with isolation buffer and then centrifuged at 16,700 × g for 10 min. A firm pellet was obtained and gently resuspended in the isolation buffer in which sucrose was substituted by 0.32 m mannitol and supplemented with 0.2 mg/ml fatty acid-free bovine serum albumin. After centrifugation at 6,900 × g for 10 min, the supernatant was rapidly decanted, and the pellet resuspended in the same buffer using a fine Teflon pestle. Protein was determined by the Folin-Lowry method using bovine serum albumin as standard (43Lowry O.H. Rosenbrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). This procedure yielded about 5 mg of protein per rat brain. Mitochondrial associated hexokinase activity was essentially present in control mitochondrial pellets (∼75%) whereas, as expected (11Bustamante E. Pedersen P.L. Biochemistry. 1980; 19: 4972-4977Crossref PubMed Scopus (55) Google Scholar), pre-incubation with Glc-6-P promoted a release of >80% of the mt-hexokinase from mitochondria. All of the experiments with isolated mitochondria were carried out at 37 °C with continuous stirring in a respiration buffer containing 10 mm Tris-HCl, pH 7.4, 0.32 m mannitol, 8 mm inorganic phosphate, 4 mm MgCl2, 0.08 mm EDTA, 1 mm EGTA, 0.2 mg/ml fatty acid-free bovine serum albumin, and 50 μm Ap5A. Determination of Hexokinase Activity—The activity of mitochondrial bound hexokinase was determined based on a previously described method with minor modifications (44Wilson J.E. Prep. Biochem. 1989; 19: 13-21PubMed Google Scholar). Briefly, mitochondrial protein used in this assay varied from 0.03-0.08 mg/ml, and the activity of hexokinase was determined by NADH formation following the absorbance at 340 nm. The assay medium contained 10 mm Tris-HCl, pH 7.4, 0.1 mm Ap5A as an inhibitor of adenylate kinase, 5 mmd-glucose, 10 mm MgCl2, 1 mm ATP, 1 mm NAD+, and 1 unit/ml Glc-6-P dehydrogenase (Leuconostoc mesenteroides) in a final volume of 1 ml. The reaction temperature was 37 °C. When ATP generated intramitochondrially by oxidative phosphorylation was used to measure the mt-hexokinase activity, the standard respiration buffer supplemented with 2 mm succinate and 0.1 mm ADP was employed. In the experiments using exogenous yeast hexokinase, the units of enzyme added were calculated considering the endogenous rat brain mt-hexokinase activity in control mitochondria in the same reaction mixture described above as a reference. The mitochondrial protein in these assays was 0.1-0.2 mg/ml. Oxygen Uptake Measurements—Oxygen uptake was measured in an oxymeter fitted with a water-jacketed Clark-type electrode (Yellow Springs Instruments Co., model 5300). Mitochondria (0.2 mg/ml) were incubated with 1.5 ml of the standard respiration buffer described above. The cuvette was closed immediately before starting the experiments. Each experiment was repeated at least three times with different mitochondrial preparations, and Fig. 1 shows representative experiments. Other additions are indicated in the Fig. 1 legend. Respiratory control ratio values were obtained with isolated mitochondria by using both pyruvate and malate as complex I substrates or succinate as a complex II substrate and were in good agreement with previous reported values (41Sims N.R. J. Neurochem. 1990; 55: 698-707Crossref PubMed Scopus (346) Google Scholar). Determination of Δψm—Mitochondrial membrane potential was measured by using the fluorescence signal of the cationic dye safranine O, which is accumulated and quenched inside energized mitochondria (45Wieckowski M.R. Wojtczak L. FEBS Lett. 1998; 423: 339-342Crossref PubMed Scopus (110) Google Scholar). Mitochondria (0.2 mg protein/ml) were incubated in the standard respiration buffer supplemented with 10 μm safranine. FCCP (5 μm) was used as a positive control to collapse Δψm. Fluorescence was detected with an excitation wavelength of 495 nm (slit 5 nm) and an emission wavelength of 586 nm (slit 5 nm) using a Hitachi (Tokyo, Japan) model F-3010 spectrofluorometer. In different laboratories it has been shown that the addition of ADP promoted a 5-10% decrease of the Δψm (37Nicholls D.G. Budd S.L. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1068) Google Scholar). Under our conditions we found that the addition of 0.2 mm ADP promoted a 20-25% decrease of the Δψm, which indicated that the effect of ADP was even more potent in our preparations than in previously reports. Data are reported as arbitrary fluorescence units. Each experiment was repeated at least three times with different mitochondrial preparations, and Fig. 4 shows representative experiments. Other additions are indicated in the Fig. 4 legend. Determination of Mitochondrial Hydrogen Peroxide Generation—Mitochondrial H2O2 production was assessed by the scopoletin oxidation method (46Boveris A. Martino E. Stoppani A.O. Anal. Biochem. 1977; 80: 145-158Crossref PubMed Scopus (111) Google Scholar). Mitochondria (0.2 mg of protein per milliliter) were incubated in the standard respiration buffer supplemented with 10 μm scopoletin and 1 unit/ml horseradish peroxidase. Fluorescence was monitored at excitation and emission wavelengths of 365 nm and 450 nm, respectively. Calibration was performed by the addition of known quantities of H2O2. Each experiment was repeated at least three times with different mitochondrial preparations, and Figs. 1, 2, and 3 show representative experiments. Other additions are indicated in the Figs. 1, 2, and 3 legends. In all experiments, small variations in the levels of H2O2 formation were observed with different preparations, but the overall pattern of response to different modulators was not changed.Fig. 3Localization of mt-hexokinase activity is important for controlling ROS production. The rate of H2O2 generation in the state 3-state 4 transition was measured in hexokinase-depleted mitochondria, which were supplemented with increasing amounts of exogenous yeast hexokinase (open circles) that used intramitochondrially generated ATP after the addition of 2 mm succinate and 0.1 mm ADP plus 5 mm glucose. The closed circle represents the rate of H2O2 generation in the state 3-state 4 transition in control mitochondria containing only endogenous hexokinase activity, which uses the intramitochondrially generated ATP after the addition of 2 mm succinate and 0.1 mm ADP plus 5 mm glucose. For the closed circle, the value shown represents mean ± S.E. of four independent preparations. The figure shows representative experiments. Similar results were obtained in at least four different independent mitochondrial preparations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cortex Cell Cultures—Cortices from 14-day-old Wistar rat embryos were dissected and cultured as described previously (47Brewer G.J. Torricelli J.R. Evege E.K. Price P.J. J. Neurosci. Res. 1993; 35: 567-576Crossref PubMed Scopus (1928) Google Scholar) with minor modifications. Cells were plated on glass coverslips coated previously with 1.5 μg/ml polyornithine in neurobasal medium supplemented with B27. After 96 h of culture, the neurobasal medium was replaced by RPMI 1640 medium, which contains 10 mm glucose supplemented with 2 μm CM-H2DCFDA (Molecular Probes) to assess intracellular ROS formation. To investigate the effects of glucose, several aliquots of the medium were supplemented with different solutions to achieve the following final concentrations: 40 mm glucose; 40 mm glucose, plus 30 mm 2-DOG; and 40 mm glucose plus 5 mm FCCP. The cells were kept for 40 min at 37 °C and 4% CO2, and randomly chosen fields were examined under a Nikon Eclipse TE300 epifluorescence microscope at a fixed exposure time. Additionally, control cells were preloaded with 1 mm NAC in neurobasal medium and kept for 45 min at 37 °C with 4% CO2. The medium was then replaced to RPMI plus 2 μm CM-H2DCFDA plus 40 mm glucose plus 1 mm NAC. Control experiments were also conducted in the presence of 10 mm glucose plus 2 μm CM-H2DCFDA plus 100 μm H2O2 in the presence or absence of 1 mm NAC. All solutions employed in cell culture experiments were prepared and kept at all times under sterile conditions. Five independent fields were counted for each experimental condition (which were carried out in triplicate). Essentially identical results were obtained in at least three repeated experiments using neurons from different animals. Determination of Intracellular Glc-6-P Content—The intracellular levels of Glc-6-P were measured by a spectrophotometric assay with minor modifications (48Lang G. Michal G. Bergmeyer H.U. Methods of Enzymatic Analysis. 3. Academic Press, New York1974: 1238-1242Google Scholar). To quantify intracellular Glc-6-P content, cultures of rat cortical neurons were kept as described above and scraped in 200 μl of 6% (v/v) trichloroacetic acid per plate (35 mm). The extract was neutralized by adding 80 μl of 1 m Tris solution. Glc-6-P levels were determined enzymatically by coupling to Glc-6-P dehydrogenase activity and monitoring this activity by NADH formation at 340 nm. Intracellular Glc-6-P levels were expressed as nmol/mg protein. Hydrogen Peroxide Formation in Control and Hexokinase-depleted Mitochondria—Most of the experiments of the present work were designed to investigate the possible involvement of mt-hexokinase activity on ROS production. To accomplish this, we first attempted to produce two different preparations of rat brain mitochondria to investigate the effects of glucose on mitochondrial metabolism in the presence or the absence of mthexokinase. In the first approach, we processed the mitochondria without any treatment (control); in the second approach, we incubated the mitochondria previously with Glc-6-P, a procedure that is known to detach mt-hexokinase from mitochondria (11Bustamante E. Pedersen P.L. Biochemistry. 1980; 19: 4972-4977Crossref PubMed Scopus (55) Google Scholar, 12Kabir F. Wilson J.E. Arch. Biochem. Biophys. 1993; 300: 641-650Crossref PubMed Scopus (36) Google Scholar, 42BeltrandelRio H. Wilson J.E. Arch. Biochem. Biophys. 1991; 286: 183-194Crossref PubMed Scopus (54) Google Scholar, 49Wilson J.E. Arch. Biochem. Biophys. 1973; 154: 332-340Crossref PubMed Scopus (25) Google Scholar). Glucose accelerated the rate of oxygen consumption in control mitochondria, whereas in mt-hexokinase-depleted mitochondria it did not affect the rate of oxygen consumption (Fig. 1, A and B). More importantly, mt-hexokinase removal did not change the respiratory control by ADP, indicating that the oxidative phosphorylation apparatus is preserved. Identical results were obtained when we utilized the complex I substrates pyruvate and malate in both mitochondrial preparations (data not shown). Control mitochondria generated H2O2 in appreciable amounts in state 4 respiration induced by succinate (Fig. 1C, trace 1) and, as expected, the addition of ADP transiently blocked the H2O2 formation during state 3 respiration (50Boveris A. Oshino N. Chance B. Biochem. J. 1972; 128: 617-630Crossref PubMed Scopus (1249) Google Scholar). Activation of mt-hexokinase by glucose substantially decreased H2O2 formation (Fig. 1C, traces 2 and 3). The mt-hexokinase-depleted mitochondria were able to generate H2O2 in state 4 respiration in a similar fashion to control mitochondria and were also sensitive to ADP (Fig. 1D, trace 4). However, the mt-hexokinase-depleted mitochondria were unresponsive to glucose (Fig. 1D, traces 2 and 3). Effect of Glucose Analogues on Hydrogen Peroxide Formation in Control Mitochondria—To support the notion that mthexokinase activity was indeed responsible for the effects described above, two different glucose analogues, 2-DOG and 6-DOG, were tested on the mitochondrial H2O2 production. 2-DOG is a substrate of hexokinase that is phosphorylated, but instead of Glc-6-P its reaction product is 2-deoxyglucose-6-P, which does not inhibit hexokinase activity in the same range as Glc-6-P (51Crane R.K. Sols A. J. Biol. Chem. 1954; 210: 597-606Abstract Full Text PDF PubMed Google Scholar, 52Chen W. Gueron M. Biochimie (Paris). 1992; 74: 867-873Crossref PubMed Scopus (90) Google Scholar). In contrast, 6-DOG is not phosphorylated by hexokinase (53Royt P.W. Biochim. Biophys. Acta. 1982; 687: 226-230Crossref PubMed Scopus (3) Google Scholar). The simultaneo
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