Chronic Treatment with Azide in Situ Leads to an Irreversible Loss of Cytochrome c Oxidase Activity via Holoenzyme Dissociation
2002; Elsevier BV; Volume: 277; Issue: 13 Linguagem: Inglês
10.1074/jbc.m112303200
ISSN1083-351X
AutoresScot C. Leary, Bruce C. Hill, C. N. Lyons, Christopher G. Carlson, Denise Michaud, Claudia S. Kraft, Kenton Ko, D. Moira Glerum, Christopher D. Moyes,
Tópico(s)Electrochemical sensors and biosensors
ResumoChronic treatment of cultured cells with very low levels of azide (I50<10 μm) leads to slow (t½ = 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azide-mediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl2, Cu-His) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either CuAor CuB. In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa3 content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties. Chronic treatment of cultured cells with very low levels of azide (I50<10 μm) leads to slow (t½ = 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azide-mediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl2, Cu-His) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either CuAor CuB. In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa3 content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties. Most ATP required by eukaryotic cells under resting conditions is generated aerobically by mitochondrial oxidative phosphorylation (OXPHOS). 1The abbreviations used are: OXPHOSoxidative phosphorylationASMCaortic smooth muscle cellsCOXcytochrome c oxidaseCScitrate synthaseCu-Hiscopper histidineETCelectron transport chainHEK293human embryonic kidney 293 cellsHShorse serumONOO−peroxynitriteO2⨪superoxide anionPBSphosphate-buffered salinePC12pheochromocytoma 12 cellsROSreactive oxygen speciesHPLChigh performance liquid chromatographySODsuperoxide dismutaseSSCstandard saline citrateDCF2,7-dichlorofluorescin Maintenance of appropriate stoichiometries of OXPHOS complexes is critical not only to ensure efficient flux of electrons through the ETC but also to minimize the potentially cytotoxic impact of mitochondrially derived ROS (1.Richter C. Gogvadze V. Laffranchi R. Schlapbach R. Schweizer M. Suter M. Walter P. Yaffee M. Biochim. Biophys. Acta. 1995; 1271: 67-74Crossref PubMed Scopus (493) Google Scholar, 2.Barrientos A. Moraes C.T. J. Biol. Chem. 1999; 274: 16188-16197Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Declines in the content of individual OXPHOS complexes frequently accompany cardiovascular and neurological diseases, as well as aging (3.Wallace D.C. Science. 1992; 256: 628-632Crossref PubMed Scopus (1112) Google Scholar, 4.Ishii N. Fujii M. Hartman P.S. Tsuda M. Yasuda K. Senoo-Matsuda N. Yanase S. Ayusawa D. Suzuki K. Nature. 1998; 394: 694-697Crossref PubMed Scopus (583) Google Scholar). oxidative phosphorylation aortic smooth muscle cells cytochrome c oxidase citrate synthase copper histidine electron transport chain human embryonic kidney 293 cells horse serum peroxynitrite superoxide anion phosphate-buffered saline pheochromocytoma 12 cells reactive oxygen species high performance liquid chromatography superoxide dismutase standard saline citrate 2,7-dichlorofluorescin The origins and consequences of the loss of a given OXPHOS complex are frequently studied in cell lines established from patients with metabolic deficiency (5.Zhu Z. Yao J. Johns T. De Fu K. Bie I. Macmillan C. Cuthbert A.P. Newbold R.F. Wang J.-c. Chevrette M. Brown G.K. Brown R.M. Shoubridge E.A. Nat. Genet. 1998; 20: 337-343Crossref PubMed Scopus (540) Google Scholar, 6.Jaksch M. Ogilvie I. Yao J. Kortenhaus G. Bresser H.-G. Gerbitz K.-D. Shoubridge E.A. Hum. Mol. Genet. 2000; 9: 795-801Crossref PubMed Scopus (195) Google Scholar, 7.Valnot I. von Kleist-Retzow J.-C. Barrientos A. Gorbatyuk M. Taanman J.-W. Mehaye B. Rustin P. Tzagoloff A. Munnich A. Rotig A. Hum. Mol. Genet. 2000; 9: 1245-1249Crossref PubMed Scopus (258) Google Scholar). However, the pathophysiological consequences of complex deficiencies have also been studied using specific inhibitors to chronically impair catalytic activity (2.Barrientos A. Moraes C.T. J. Biol. Chem. 1999; 274: 16188-16197Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 8.Betarbet R. Sherer T.B. MacKenzie G. Garcia-Osuna M. Panov A.V. Greenamyre J.T. Nat. Neurosci. 2000; 3: 1301-1306Crossref PubMed Scopus (2957) Google Scholar). Sodium azide is commonly used in vitro as a rapid and reversible inhibitor of COX, the terminal enzyme of the ETC that catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen (9.Yoshikawa S. Caughey W. J. Biol. Chem. 1992; 267: 9757-9766Abstract Full Text PDF PubMed Google Scholar, 10.Li W. Palmer G. Biochemistry. 1993; 32: 1833-18436Crossref PubMed Scopus (58) Google Scholar, 11.Yoshikawa S. Shinzawa-Itoh K. Nakashima R. Yaono R. Yamashita E. Inoue N. Yao M. Fei M.J. Libeu C.P. Mizushima T. Yamaguchi H. Tomizaki T. Tsukihara T. Science. 1998; 280: 1723-1729Crossref PubMed Scopus (973) Google Scholar). The ability of azide to rapidly bind to the binuclear center, thereby inhibiting COX activity, has led to its frequent use in acute studies, including those designed to address the role of mitochondrial oxygen sensing in signal transduction (12.Budinger G.R.S. Duranteau J. Chandel N.S. Schumacker P.T. J. Biol. Chem. 1998; 273: 3320-3326Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 13.Chandel N.S. Maltepe E. Goldwasser E. Mathieu C.E. Simon M.C. Schumacker P.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 95: 11715-11720Crossref Scopus (1602) Google Scholar). We previously reported that chronic treatment of cultured myocytes with micromolar levels of azide results in the irreversible inhibition of COX activity (14.Leary S.C. Battersby B.J. Hansford R.G. Moyes C.D. Biochim. Biophys. Acta. 1998; 1365: 522-530Crossref PubMed Scopus (84) Google Scholar). This mode of inhibition clearly differs from the classic mechanism of azide-mediated COX inhibition in that it is of higher affinity (0.01–0.1versus 1–10 mm) and irreversible in nature. The mechanism(s) by which azide mediates these effects is unknown. Moreover, its potential relevance to COX deficiency observed in a more physiologically relevant context has not been evaluated despite previous reports of selective COX losses leading to Alzheimer's disease-like symptoms in rats that had been chronically infused with sodium azide (15.Bennett M.C. Mlady G.W. Kwon Y.-H. Rose G.M. J. Neurochem. 1996; 66: 2606-2611Crossref PubMed Scopus (67) Google Scholar, 16.Berndt J.D. Callaway N.L. Gonzalez-Lima F. J. Toxicol. Environ. Health. 2001; 63: 67-77Crossref Scopus (36) Google Scholar). Several plausible mechanisms exist that could account for the azide-mediated, irreversible inhibition of COX activity. First, the capacity of azide to act as a chelator of first order transition series metals could affect COX activity by either terminal binding to or stripping of one or both copper centers from highly conserved domains within mitochondrially encoded subunits I and II (CuA on COX II, CuB on COX I; Refs. 11.Yoshikawa S. Shinzawa-Itoh K. Nakashima R. Yaono R. Yamashita E. Inoue N. Yao M. Fei M.J. Libeu C.P. Mizushima T. Yamaguchi H. Tomizaki T. Tsukihara T. Science. 1998; 280: 1723-1729Crossref PubMed Scopus (973) Google Scholar and 17.Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (520) Google Scholar). Second, because copper metabolism is critical to the maturation and assembly of individual COX subunits into a functional holoenzyme complex (18.Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 19.Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 20.Hiser L. Di Valentin M. Hamer A.G. Hosler J.P. J. Biol. Chem. 2000; 275: 619-623Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), azide may impair either its delivery or insertion into the complex. Third, azide may act as a suicide metabolite to inhibit COX activity during enzyme turnover in a manner analogous to azide-mediated loss of catalase activity (21.Lardinois O.M. Rouxhet P.G. Biochim. Biophys. Acta. 1996; 1298: 180-190Crossref PubMed Scopus (30) Google Scholar). Accordingly, it has recently been shown that the COX-mediated conversion of azide to the azidyl radical leads to its irreversible inactivation in vitro, although this mechanism requires very high concentrations of both azide and H2O2 (22.Chen Y.-R. Sturgeon B.E. Gunther M.R. Mason R.P. J. Biol. Chem. 1999; 274: 24611-24616Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Fourth, chronic azide effects on COX activity may arise indirectly from elevated bulk phase production of ROS since azide is also a potent inhibitor of SOD (23.Misra H.P. Fridovich I. Arch. Biochem. Biophys. 1978; 189: 317-322Crossref PubMed Scopus (127) Google Scholar) and catalase (21.Lardinois O.M. Rouxhet P.G. Biochim. Biophys. Acta. 1996; 1298: 180-190Crossref PubMed Scopus (30) Google Scholar, 24.Ghadermarzi M. Moosavi-Movahedi A.A. Biochim. Biophys. Acta. 1999; 1431: 30-36Crossref PubMed Scopus (25) Google Scholar) activities. In the present study, we systematically addressed these possibilities to clarify the mechanism(s) by which azide irreversibly inhibits COX activity. Collectively, our studies suggest that azide treatment results in a loss of catalytic activity by accelerating the rate of holoenzyme dissociation, possibly through interactions with the structure or coordination of heme moieties. All culture media, sera, and antibiotics were purchased from Invitrogen. C2C12 cells were grown in proliferation medium consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. At 70% confluency, myoblasts were switched to differentiation medium consisting of Dulbecco's modified Eagle's medium supplemented with 2% HS. Sol8, HEK293, and ASMC were cultured in proliferation medium supplemented with 10% fetal bovine serum, whereas PC12 cells were maintained in proliferation medium containing 20% fetal bovine serum. Penicillin, streptomycin, and neomycin were included in all media. The medium was changed every 2–3 days in proliferating and serum-starved cells. Assays for all enzymes were optimized to ensure that neither substrates nor co-factors were limiting. The activities of NADH ubiquinone oxidoreductase (EC 1.6.5.3), COX (EC 1.9.3.1), CS (EC4.1.3.7) (25.Moyes C.D. Mathieu-Costello O.A. Tsuchiya N. Filburn C. Hansford R.G. Am. J. Physiol. 1997; 272: C1345-C1351Crossref PubMed Google Scholar), glutathione peroxidase (EC 1.11.1.9) (26.Flohe L. Gunzler W.A. Methods Enzymol. 1984; 105: 114-121Crossref PubMed Scopus (4070) Google Scholar), and catalase (EC 1.11.1.6) (27.Aebi H. Bergmeyer H.U. Methods of Enzymatic Analysis. Academic Press, New York1974: 673-678Crossref Google Scholar) were measured from whole cell extracts as previously described. The activities of individual ETC enzymes were also assayed using isolated mitochondria (28.Bindoff L.A. Birch-Machin M.A. Cartlidge N.E. Parker Jr., W.D. Turnbull D.M. J. Neurol. Sci. 1991; 104: 203-208Abstract Full Text PDF PubMed Scopus (194) Google Scholar). The complex V (EC 3.6.1.34) assay contained 5 mm MgCl2, 100 mm KCl, 1 mm phosphoenolpyruvate, 5 mm ATP, 0.15 mm NADH, and 1 unit each lactate dehydrogenase and pyruvate kinase in 50 mm HEPES (pH 7.4) in the presence or absence of saturating amounts of oligomycin (0.24–0.72 μg). No NADH oxidation was observed in the absence of mitochondria. Total cellular SOD (EC 1.15.1.1) was assayed at 550 nm using the aerobic xanthine/xanthine oxidase system (23.Misra H.P. Fridovich I. Arch. Biochem. Biophys. 1978; 189: 317-322Crossref PubMed Scopus (127) Google Scholar, 29.Crapo J.D. McCord J.M. Fridovich I. Methods Enzymol. 1978; 53: 382-393Crossref PubMed Scopus (618) Google Scholar). MnSOD activity was measured from mitochondrial extracts in the presence of 5 mm KCN to inhibit any contaminating CuZn SOD. Protocols for measuring cellular protein content, lactate concentration in the culture media, and whole cell respiration were as previously described (16.Berndt J.D. Callaway N.L. Gonzalez-Lima F. J. Toxicol. Environ. Health. 2001; 63: 67-77Crossref Scopus (36) Google Scholar). Subconfluent myoblasts were grown in 10% fetal bovine serum on poly-l-lysine-treated glass coverslips. Coverslips were fitted to a 260-μl perfusion chamber thermostatted to 37 °C (Warner Instruments) on a Zeiss fluorescent microscope stage, superfused for 35 min with PBS supplemented with 25 mm glucose, 4 mm glutamine, 1 mmpyruvate, 1.8 mm CaCl2, 0.8 mmMgSO4, and 1% penicillin-streptomycin-neomycin containing 5 μm 2,7-dichlorofluorescin (DCF) diacetate (Molecular Probes). Fluorescence was measured (20× magnification, 100-ms exposure, 4 × 4 binning) using a CCD camera (Cooke Sensicam) and optical filters appropriate for DCF fluorescence (excitation peak, 490 nm; emission peak, 526 nm). The same cells were subsequently perfused for 5 min with 5 mmH2O2 to obtain maximal fluorescence (30.Narayan P. Mentzer Jr., R.M. Lasley R.D. J. Mol. Cell. Cardiol. 2001; 33: 121-129Abstract Full Text PDF PubMed Scopus (78) Google Scholar). The mean pixel intensities of cells and background were determined using Slidebook (Intelligent Imaging Innovations). Because of potential excitation-dependent oxidation of DCFH, only one field of view was analyzed on each coverslip. Plates were rinsed and harvested in ice-cold PBS. Pelleted cells were flash-frozen in liquid nitrogen and stored at −80 °C for at least 24 h. Thawed cells pellets were resuspended in ice-cold isolation buffer (250 mm sucrose, 20 mm HEPES (pH 7.4), and 1 mm EDTA) supplemented with a protease inhibitor mixture (Sigma) and homogenized with 10 passes through a pre-chilled, zero clearance homogenizer (Kontes Glass Co.). Cell debris and nuclei were pelleted with 2 10-min spins at 650 × g. The resultant supernatant (S1) was spun for 15 min at 14,000 × g to collect the mitochondrial fraction. Denatured mitochondrial and S1 fractions (10 μg) were resolved using a 12% SDS-PAGE gel (31.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar), and electroblotted (Bio-Rad, Mini-protean system) onto nitrocellulose membranes (Zymotech Inc.). Rabbit polyclonal antibodies directed against Cu/Zn and MnSOD (StressGen Biotechnologies Corp.) and cytochrome c (Santa Cruz Biotechnologies) and mouse monoclonal antibodies for anti-COX I, COX IV, and ND1 (Molecular Probes) were diluted to 1:1,000. Polyclonal anti-COX II (Dr. T. L. Mason, University of Massachusetts), anti-actin (Sigma), and anti-catalase (Rockland Immunochemicals Inc.) antibodies were diluted to 1:40, 1:5,000, and 1:25,000, respectively. Mouse anti-39-kDa (Molecular Probes) and human anti-porin (Calbiochem) monoclonal antibodies were diluted to 1:2,000. COX I and IV antibodies were resuspended in PBS containing 1% bovine serum albumin and 0.1% Tween 20. All other antibodies were reconstituted in Tris-buffered saline containing 0.05% Tween 20 supplemented with 2% low fat skim milk powder. Membranes were blocked for 1 h at room temperature in Tris-buffered saline/Tween supplemented with 5% low fat skim milk and incubated overnight at 4 °C in the primary antibody of interest. After a 1-h incubation at room temperature with either secondary anti-mouse (Pierce, 1:10,000) or anti-rabbit (Promega, 1:2,500) horseradish peroxidase antibodies, immunoreactive proteins were detected by luminol-enhanced chemiluminescence (Pierce). Blue native PAGE (32.Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1909) Google Scholar, 33.Schägger H. Bentlage H. Ruitenbeck W. Pfeiffer K. Rother C. Böttcher-Purkl A. Lodemann E. Electrophoresis. 1995; 16: 763-770Crossref PubMed Scopus (71) Google Scholar) was performed on mitoplasts that were isolated using a 1.2:1 digitonin to protein ratio and solubilized (1.5 mg/ml) on ice for 30 min in 750 mm 6-aminocaproic acid, 50 mm Bistris (pH 7.0), 1.5 mm EDTA, and 1.5% w/v laurylmaltoside (34.Klement P. Nijtmans L.G.J. Van den Bogert C. Houstek J. Anal. Biochem. 1995; 231: 218-224Crossref PubMed Scopus (94) Google Scholar). Samples were centrifuged for 20 min at 20,000 × g, and the relative distribution of complex I activity between the pellet and the extract was measured as an index of solubilization of inner membrane proteins (extract typically contained 80–90% of the total activity). Loading dye was added to each sample at a 1:4 ratio of Coomassie:laurylmaltoside, and equal units of complex I were loaded in each lane of a 6–15% continuous gradient gel. Protocols used for in-gel assays (35.Zerbetto E. Vergani L. Dabbeni-Sala F. Electrophoresis. 1997; 18: 2059-2064Crossref PubMed Scopus (254) Google Scholar), immunoblotting of first-dimension gels and second dimension electrophoresis (36.Dekker P.J.T. Martin F. Maarse A.C. Bömer U. Müller H. Guiard B. Meijer M. Rassow J. Pfanner N. EMBO J. 1997; 16: 5408-5419Crossref PubMed Scopus (238) Google Scholar) were as previously described. NADH ubiquinone oxidoreductase and COX holoenzyme levels from first-dimension immunoblots were quantified using an enhanced chemifluorescence detection system (Amersham Biosciences, Inc.) according to the manufacturers' specifications. Mitochondria (2.5 mg/ml) were solubilized on ice for 1 h in 750 mm 6-aminocaproic acid, 50 mm Bistris (pH 7.0) containing 1% w/v Triton X-100. Samples were centrifuged for 10 min at 14,000 × g to remove debris. UV-visible spectra were recorded on an OLIS-refurbished Aminco DW-2 UV/VIS spectrophotometer at room temperature and are presented as reduced-oxidized differences. The oxidized state is taken as the form of the sample in air and is unchanged upon the addition of ferricyanide. The reduced state is generated by addition of a few grains of solid sodium dithionite to the air-oxidized sample. Total heme A content from 3 mg of mitochondrial protein was extracted and measured by reverse phase HPLC as previously described (37.Barros M.H. Carlson C.G. Glerum D.M. Tzagoloff A. FEBS Lett. 2001; 492: 133-138Crossref PubMed Scopus (119) Google Scholar). Total RNA was purified from guanidinium thiocyanate extracts using a standard acid phenol protocol (38.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63184) Google Scholar). Denatured RNA (10 μg) was fractionated using a 1% agarose-formaldehyde gel system. A cDNA for the muscle-specific isoform of COX VIa was amplified from rat soleus reverse-transcriptase template at 60 °C with 5′-ctgacctttgtgctggctct-3′ and 5′-gattgacgtggggattgtg-3′ using standard PCR conditions, cloned into pCR 2.1 (Invitrogen), and sequenced. All other probes for mtDNA- and nuclear-encoded mRNA species were obtained as previously described (16.Berndt J.D. Callaway N.L. Gonzalez-Lima F. J. Toxicol. Environ. Health. 2001; 63: 67-77Crossref Scopus (36) Google Scholar, 25.Moyes C.D. Mathieu-Costello O.A. Tsuchiya N. Filburn C. Hansford R.G. Am. J. Physiol. 1997; 272: C1345-C1351Crossref PubMed Google Scholar). Membranes were prehybridized (3 h) and hybridized (12–18 h) at 65 °C in modified Church's buffer (0.5 m sodium phosphate (pH 7.0), 7% SDS, and 10 mm EDTA). Membranes were washed twice at room temperature for 15 min with 2× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate), 0.1% SDS and twice at 50 °C for 15 min with 0.1× SSC, 0.1% SDS. Blots were visualized by phosphorimaging, and relative signal strength was quantified using ImageQuant software (Molecular Dynamics). Differences in loading across lanes were normalized using a probe for α-tubulin mRNA. To pulse label mitochondrial translation products (39.Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar), myotubes grown in 60-mm plates were washed twice with sterile PBS and incubated for 30 min in pre-warmed, methionine-free Dulbecco's modified Eagle's medium supplemented with 2% dialyzed HS. After an additional 5-min incubation in the presence of 100 μg/ml emetine, cells were incubated with 400 μCi of Expre35S35S (Mandel) for 1 h. Excess label was chased by the addition of regular Dulbecco's modified Eagle's medium, 2% HS for 10 min. Cells were washed 3 times with PBS and harvested by trypsinization. Samples (10 μg) were sonicated on ice and fractionated on a 12–20% gradient gel at 7 mA for 14–16 h. The gel was then fixed for 1 h and dried for autoradiography. Significant differences (p < 0.05) between control and treated groups for all measured parameters were detected using one-way analysis of variance and identified post hoc using the Tukey-Kramer honestly signficantly different. Chronic treatment of C2C12 myotubes with micromolar amounts of azide caused a time-dependent loss of COX activity (data not shown; Ref. 14.Leary S.C. Battersby B.J. Hansford R.G. Moyes C.D. Biochim. Biophys. Acta. 1998; 1365: 522-530Crossref PubMed Scopus (84) Google Scholar), a resultant decline in whole cell respiration, and a concomitant increase in the rate of intracellular lactate accumulation (Fig. 1A). The azide effect on COX activity was also observed in three other cell backgrounds (HEK293, PC12, and ASMC), with a 24-h treatment with 100 μm azide, resulting in the inhibition of 65–90% of total activity (Fig. 1B). Loss of catalytic activity was irreversible, both in situ and in vitro. C2C12 myotubes pulsed for 6 h with 100 μm azide continued to lose COX activity 24 h after the removal of azide from the culture media (6 versus24 h, 49 versus 33% of control). Azide-treated C2C12 cell extracts dialyzed for 24 h (50 mm Tris (pH 8.0), 4 °C) in the presence or absence of reductant (β-mercaptoethanol, dithiothreitol) also failed to recover catalytic activity (data not shown). To determine whether azide required access to the binuclear center to irreversibly inhibit COX activity in situ, C2C12 myotubes were co-incubated with equimolar amounts of azide and cyanide (10 μm each). Despite a much higher relative affinity of cyanide (Ki 1 versus 64 μm; Refs. 10.Li W. Palmer G. Biochemistry. 1993; 32: 1833-18436Crossref PubMed Scopus (58) Google Scholar and 41.Tsubaki M. Yoshikawa S. Biochemistry. 1993; 32: 174-182Crossref PubMed Scopus (42) Google Scholar) and its ability to competitively displace bound azide from the binuclear center in vitro (42.Partridge R.S. Monroe S.M. Parks J.K. Johnson K. Parker Jr., W.D. Eaton G.R. Eaton S.S. Arch. Biochem. Biophys. 1994; 310: 210-217Crossref PubMed Scopus (56) Google Scholar), it neither attenuated nor abrogated azide-mediated COX inhibition (expressed as % of control: azide, 51.1; KCN, 102.5; azide + KCN, 47.1). Collectively, these data argue against azide exerting its in situ effects on COX activity via classically described ligand interactions with the binuclear center (see Refs. 9.Yoshikawa S. Caughey W. J. Biol. Chem. 1992; 267: 9757-9766Abstract Full Text PDF PubMed Google Scholar and 10.Li W. Palmer G. Biochemistry. 1993; 32: 1833-18436Crossref PubMed Scopus (58) Google Scholar). The irreversible loss of catalytic activity could not be mimicked by dialyzing extracts of untreated C2C12 myotubes, rat skeletal muscle, or purified bovine COX against 0–100 μm azide (24 h in 50 mm Tris (pH 8.0), 4 °C; data not shown). Given that azide-induced losses in catalytic activity only occurred in situ, we investigated the potential requirement of an intact ETC to the observed azide effects (see Ref. 42.Partridge R.S. Monroe S.M. Parks J.K. Johnson K. Parker Jr., W.D. Eaton G.R. Eaton S.S. Arch. Biochem. Biophys. 1994; 310: 210-217Crossref PubMed Scopus (56) Google Scholar). Previous reports that azide is a potent inhibitor of the ATPase activity of complex V (43.Kobayashi H. Maeda M. Anraku Y. J. Biochem. (Tokyo). 1977; 81: 1071-1077Crossref PubMed Scopus (22) Google Scholar,44.Harris D.A. Biochim. Biophys. Acta. 1989; 974: 156-162Crossref PubMed Scopus (41) Google Scholar) also prompted us to evaluate its effects on the activity of a spectrum of other mitochondrial enzymes. Although azide treatment resulted in a significant loss of COX activity, it had no effect on a Krebs cycle enzyme (CS), individual complexes of the ETC, and the ATPase component of complex V (Fig. 1C). Incubation of C2C12 cells for 24 h with specific inhibitors of complexes I (2–200 nm rotenone) and III (2 μg/ml antimycin A, 0.1–10 μm myxothiazol) in the presence or absence of azide neither caused a specific COX loss nor abrogated azide-mediated COX losses (data not shown). Although 24-h incubations with oligomycin (2.4 μg/ml), a complex V inhibitor, enhanced the specific loss of COX activity mediated by azide (Table I), marked inhibition of 35S incorporation into mitochondrial translation products (36.2% of control; data not shown) suggested that the oligomycin effect was attributable to reduced availability of mitochondrially encoded COX subunits required for holoenzyme assembly.Table IAzide and oligomycin effects on enzyme activityTreatmentEnzyme activityCOXNDHCatalaseTotal SOD% control10 μm azide53.2 ± 4.6aDenotes a significant difference (p < 0.05) between control and treated cells.112.3 ± 6.974.4 ± 4.8aDenotes a significant difference (p < 0.05) between control and treated cells.63.5 ± 4.8aDenotes a significant difference (p < 0.05) between control and treated cells.2.4 μg/ml oligomycin91.5 ± 1.8128.7 ± 4.2aDenotes a significant difference (p < 0.05) between control and treated cells.123.7 ± 4.9aDenotes a significant difference (p < 0.05) between control and treated cells.91.5 ± 8.210 μm azide + 2.4 μg/ml oligomycin26.9 ± 5.1aDenotes a significant difference (p < 0.05) between control and treated cells.bDenotes a significant difference (p < 0.05) between cells treated with azide alone versus azide plus oligomycin.147.9 ± 20.4aDenotes a significant difference (p < 0.05) between control and treated cells.bDenotes a significant difference (p < 0.05) between cells treated with azide alone versus azide plus oligomycin.107.8 ± 7.3bDenotes a significant difference (p < 0.05) between cells treated with azide alone versus azide plus oligomycin.38.2 ± 3.8aDenotes a significant difference (p < 0.05) between control and treated cells.bDenotes a significant difference (p < 0.05) between cells treated with azide alone versus azide plus oligomycin.C2C12 cells were differentiated for 6 days under serum-starved conditions and treated for 24 h with either 10 μmazide or 2.4 μg/ml oligomycin alone or in combination. COX, NADH ubiquinone oxidoreductase (NDH), catalase, and total SOD activities were assayed at 37 °C from solubilized cell extracts as outlined under "Experimental Procedures." All rates are expressed relative to controls (n = 5).a Denotes a significant difference (p < 0.05) between control and treated cells.b Denotes a significant difference (p < 0.05) between cells treated with azide alone versus azide plus oligomycin. Open table in a new tab C2C12 cells were differentiated for 6 days under serum-starved conditions and treated for 24 h with either 10 μmazide or 2.4 μg/ml oligomycin alone or in combination. COX, NADH ubiquinone oxidoreductase (NDH), catalase, and total SOD activities were assayed at 37 °C from solubilized cell extracts as outlined under "Experimental Procedures." All rates are expressed relative to controls (n = 5). Although other bioenergetic enzymes were unaffected by azide treatment, marked inhibition of CuZn SOD (12.0 ± 3.6% of control) and catalase (30.8 ± 1.3% of control) was also observed (Fig. 1D). This suggested the possibility that altered cellular capacity to metabolize ROS may contribute to the loss of COX activity. However, my
Referência(s)