Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase Plays a Major Role in Preventing Oxidative Injury to Cells
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.4924
ISSN1083-351X
AutoresMasayoshi Arai, Hirotaka Imai, Tomoko Koumura, Madoka Yoshida, Kazuo Emoto, Masato Umeda, Nobuyoshi Chiba, Yasuhito Nakagawa,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoPhospholipid hydroperoxide glutathione peroxidase (PHGPx) is synthesized as a long form (L-form; 23 kDa) and a short form (S-form; 20 kDa). The L-form contains a leader sequence that is required for transport to mitochondria, whereas the S-form lacks the leader sequence. A construct encoding the leader sequence of PHGPx tagged with green fluorescent protein was used to transfect RBL-2H3 cells, and the fusion protein was transported to mitochondria. The L-form of PHGPx was identified as the mitochondrial form of PHGPx and the S-form as the non-mitochondrial form of PHGPx since preferential enrichment of mitochondria for PHGPx was detected in M15 cells that overexpressed theL-form of PHGPx, whereas no similar enrichment was detected in L9 cells that overexpressed the S-form. Cell death caused by mitochondrial injury due to potassium cyanide (KCN) or rotenone (chemical hypoxia) was considerably suppressed in the M15 cells, whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected with the vector alone) succumbed to the cytotoxic effects of KCN. Flow cytometric analysis showed that mitochondrial PHGPx suppressed the generation of hydroperoxide, the loss of mitochondrial membrane potential, and the loss of plasma membrane integrity that are induced by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial functions and cell death by reducing intracellular hydroperoxides. Mitochondrial PHGPx failed to protect M15 cells from mitochondrial injury by carbonyl cyanide m-chlorophenylhydrazone, which directly reduces membrane potential without the generation of hydroperoxides. M15 cells were more resistant than L9 cells to cell death caused by direct damage to mitochondria and to extracellular oxidative stress. L9 cells were more resistant totert-butylhydroperoxide than S1 cells, whereas resistance to t-butylhydroperoxide was even more pronounced in M15 cells than in L9 cells. These results suggest that mitochondria might be a target for intracellular and extracellular oxidative stress and that mitochondrial PHGPx, as distinct form non-mitochondrial PHGPx, might play a primary role in protecting cells from oxidative stress. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is synthesized as a long form (L-form; 23 kDa) and a short form (S-form; 20 kDa). The L-form contains a leader sequence that is required for transport to mitochondria, whereas the S-form lacks the leader sequence. A construct encoding the leader sequence of PHGPx tagged with green fluorescent protein was used to transfect RBL-2H3 cells, and the fusion protein was transported to mitochondria. The L-form of PHGPx was identified as the mitochondrial form of PHGPx and the S-form as the non-mitochondrial form of PHGPx since preferential enrichment of mitochondria for PHGPx was detected in M15 cells that overexpressed theL-form of PHGPx, whereas no similar enrichment was detected in L9 cells that overexpressed the S-form. Cell death caused by mitochondrial injury due to potassium cyanide (KCN) or rotenone (chemical hypoxia) was considerably suppressed in the M15 cells, whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected with the vector alone) succumbed to the cytotoxic effects of KCN. Flow cytometric analysis showed that mitochondrial PHGPx suppressed the generation of hydroperoxide, the loss of mitochondrial membrane potential, and the loss of plasma membrane integrity that are induced by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial functions and cell death by reducing intracellular hydroperoxides. Mitochondrial PHGPx failed to protect M15 cells from mitochondrial injury by carbonyl cyanide m-chlorophenylhydrazone, which directly reduces membrane potential without the generation of hydroperoxides. M15 cells were more resistant than L9 cells to cell death caused by direct damage to mitochondria and to extracellular oxidative stress. L9 cells were more resistant totert-butylhydroperoxide than S1 cells, whereas resistance to t-butylhydroperoxide was even more pronounced in M15 cells than in L9 cells. These results suggest that mitochondria might be a target for intracellular and extracellular oxidative stress and that mitochondrial PHGPx, as distinct form non-mitochondrial PHGPx, might play a primary role in protecting cells from oxidative stress. Mitochondria are a major physiological source of reactive oxygen species (ROS), 1The abbreviations ROSreactive oxygen speciesBSObuthionine sulfoximineCCCPcarbonyl cyanidem-chlorophenylhydrazonecGPxcytosolic glutathione peroxidaseDCFH-DA5,6-carboxy-2′,7′-dichlorofluorescein diacetateGFPgreen fluorescent proteinLDHlactate dehydrogenasePBSphosphate-buffered salinePHGPxphospholipid hydroperoxide glutathione peroxidasePIpropidium iodideRBLrat basophile leukemia cellsRh123rhodamine 123t-BuOOHtert-butylhydroperoxideTNF-αtumor necrosis factor-αSODsuperoxide dismutaseBSAbovine serum albumin which can be generated during mitochondrial respiration (1Guarnieri C. Muscari C. Caldarera C.M. Emerit I. Chance B. Free Radicals and Aging. Birkhauser Verlag, Basel1992: 73-77Crossref Scopus (21) Google Scholar). Superoxide radicals, formed by minor side reactions of the mitochondrial electron transport chain or by an NADH-independent enzyme, can be converted to H2O2 and to the powerful oxidant, the hydroxyl radical (2Nohl H. FEBS Lett. 1987; 214: 269-273Crossref PubMed Scopus (100) Google Scholar). Thus, mitochondria are continually exposed to ROS that cause peroxidation of membrane lipids, cleavage of mitochondrial DNA, and impairment of ATP generation, with resultant irreversible damage to mitochondria. Mitochondrial dysfunction might contribute to the pathogenesis of various human neurodegenerative disorders, such as Parkinson's, Alzheimer's, and Huntington's diseases, amyotrophic lateral sclerosis, stroke, epilepsy, aging, and the AIDS dementia complex (3Shigenaga M.K. Hagen T.M. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10771-10778Crossref PubMed Scopus (1848) Google Scholar, 4Lestienne P. Bataille N. Biomed. Pharmacother. 1994; 48: 199-214Crossref PubMed Scopus (43) Google Scholar, 5Tritschler H.J. Packer L. Medori R. Biochem. Mol. Biol. Int. 1994; 34: 169-181PubMed Google Scholar). However, ROS don't have exclusively toxic effects; low levels of ROS generated in mitochondria can act as signaling molecules under physiological conditions. ROS produced in mitochondria can activate transcription factors, such as NFκB and AP-1 (6Rao G.N. Glasgow W.C. Eling T.E. Runge M.S. J. Biol. Chem. 1996; 271: 27760-27764Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), and can function as signals in apoptosis that is induced by TNF-α (7Donato N.J. Perez M. J. Biol. Chem. 1998; 273: 5067-5072Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), ceramide (8Quillet-Mary A. Jaffrezou J.-P. Mansat V. Bordier C. Naval J. Laurent G. J. Biol. Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (446) Google Scholar), and chemical hypoxia (9Shimizu S. Eguchi Y. Kamiike W. Matsuda H. Tsujimoto Y. Oncogene. 1996; 12: 2251-2257PubMed Google Scholar). reactive oxygen species buthionine sulfoximine carbonyl cyanidem-chlorophenylhydrazone cytosolic glutathione peroxidase 5,6-carboxy-2′,7′-dichlorofluorescein diacetate green fluorescent protein lactate dehydrogenase phosphate-buffered saline phospholipid hydroperoxide glutathione peroxidase propidium iodide rat basophile leukemia cells rhodamine 123 tert-butylhydroperoxide tumor necrosis factor-α superoxide dismutase bovine serum albumin The production of ROS in mitochondria is strictly regulated by mitochondrial antioxidant enzymes that include phospholipid hydroperoxide glutathione peroxidase (PHGPx), classical glutathione peroxidase (cGPx), and Mn-superoxide dismutase (Mn-SOD). The importance of antioxidant enzymes in mitochondria is indicated by the fact that knock-out mice without a gene for Mn-SOD suffer from catastrophic effects (10Li Y. Huang T.T. Carlson E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger C. Chan P.H. Wallace D.C. Epstein C.J. Nat. Genet. 1995; 11: 376-381Crossref PubMed Scopus (1462) Google Scholar). By contrast, knock-out mice without a gene for cGPx are quite vigorous. Some of these tissues remain very resistant to oxidative stress even though GPx is the only antioxidant enzyme that is known to reduce the H2O2 produced by Mn-SOD in mitochondria since mitochondria in most mammalian cells lack catalase activity (11Esworthy R.S. Ho Y.S. Chu F.F. Arch. Biochem. Biophys. 1997; 340: 59-63Crossref PubMed Scopus (134) Google Scholar). Two types of GPx, namely cGPx and PHGPx, are located in mitochondria. PHGPx is the only known intracellular antioxidant enzyme that can directly reduce peroxidized phospholipids (12Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-72Crossref PubMed Scopus (766) Google Scholar) and cholesterol (13Thomas J.P. Maiorino M. Ursini F. Girotti A.W. J. Biol. Chem. 1990; 265: 454-461Abstract Full Text PDF PubMed Google Scholar) in membranes. Therefore, PHGPx that can reduce H2O2, rather than cGPx, is thought to contribute to the enzymatic defenses against oxidative damage to mitochondria (14Flohé L. Dolphin D. Poulson R. Avramovic O. Glutathione: Chemical, Biochemical, and Medical Aspects. John Wiley & Sons, Inc., New York1989: 643-731Google Scholar). However, the PHGPx in mitochondria has not been fully characterized. We previously cloned a cDNA for PHGPx from the rat (15Imai H. Sumi D. Hanamoto A. Arai M. Sugiyama A. Chiba N. Kuchino Y. Nakagawa Y. J. Biochem. (Tokyo). 1995; 118: 1061-1067Crossref PubMed Scopus (59) Google Scholar, 16Arai M. Imai H. Sumi D. Imanaka T. Takano T. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 227: 433-439Crossref PubMed Scopus (97) Google Scholar) and demonstrated that a short 20-kDa (S-form) and a long 23-kDa (L-form) form of PHGPx were translated from the cDNA, which included two potential sites for the initiation of translation in vitro(16Arai M. Imai H. Sumi D. Imanaka T. Takano T. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 227: 433-439Crossref PubMed Scopus (97) Google Scholar). We showed that the L-form included a leader sequence and was selectively imported into the mitochondria of rat liver by an import system in vitro (16Arai M. Imai H. Sumi D. Imanaka T. Takano T. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 227: 433-439Crossref PubMed Scopus (97) Google Scholar). Stable transformants of rat basophile leukemia 2H3 (RBL-2H3) cells, in which the S-form of PHGPx was overexpressed, were resistant to the cell death caused by a radical initiator or oxidized lipids (17Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). The S-form of PHGPx markedly inhibited the production of leukotrienes by 5-lipoxygenase by preventing production of intracellular hydroperoxides around the nucleus (18Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In the present study, RBL-2H3 cells that overexpressed theL-form of PHGPx were established and compared with those that overexpressed the S-form in an attempt to estimate the functional roles of the two types of PHGPx in protection against intracellular and extracellular oxidative stress. The L-form of PHGPx was more effective than the S-form in preventing cell death that was caused by ROS generated in mitochondria and by exogenously added hydroperoxides. Antibodies against PHGPx and cGPx were prepared as described previously (17Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). Monoclonal antibodies against histone H1 and125I-protein A (2.60–3.70 TBq/g) were purchased from Cosmobio Co. Ltd. (Tokyo, Japan) and ICN Biochemicals Inc. (Irvine, CA), respectively. Rhodamine 123 (Rh123), 5,6-carboxy-2′,7′-dichlorofluorescein-diacetate (DCFH-DA), andcis-parinaric acid were obtained from Funakoshi Co. Ltd. (Tokyo, Japan). Ammonium 7-fluoroben-2-oxa-1,3-diazo-4-sulfonate (SBD-F) and tri-n-butyl phosphine were obtained from Wako Co. Ltd. (Tokyo, Japan). Propidium iodide (PI) and monoclonal antibodies against cytochrome oxidase subunit IV was obtained from Molecular Probes (Leiden, Netherlands). KCN, rotenone, CCCP, oligomycin, and BSO were purchased from Sigma. A BamHI fragment of pRPHGPx4 (16Arai M. Imai H. Sumi D. Imanaka T. Takano T. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 227: 433-439Crossref PubMed Scopus (97) Google Scholar) was subcloned into pSRα, as the expression vector, to construct pSRα-L-form PHGPx that encoded theL-form of PHGPx (19Takebe Y. Seiki M. Fujisawa J. Hoy P. Yokota K. Arai K. Yoshida M. Arai N. Mol. Cell. Biol. 1988; 8: 466-472Crossref PubMed Google Scholar). S-probe and L-probe were made from pRPHGPx4 by polymerase chain reactions for construction of S-GFP and L-GFP. The primers for construction of the S-probe, in which the cDNA encoded the 42 amino acids from the first residue of the S-form of PHGPx, were 5′-ACATAAGCTTGCTGGCACCATGTGTGCA-3′ and 5′-ATTAGGTACCGGCCACGTTGGTGACGAT-3′. The primers for construction of the L-probe, in which the cDNA encoded the 32 amino acids from the first residue of the L-form of PHGPx, were 5′-ATTTAAGCTTCCGGCCGCCGAGATGAGC-3′ and 5′-ATTAGGTACCGCGGGATGCACACATGGT-3′. The BamHI andKpnI fragments of S-probe and L-probe were inserted between the BamHI and KpnI sites of the GFP expression vector (pCMX-SAP/Y145 F) that had been constructed by Ogawa et al. (20Ogawa H. Inouye S. Tsuji FL. Yasuda K. Umesono K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11899-11903Crossref PubMed Scopus (235) Google Scholar). We used the previously established control line of cells (S1 cells) and L9 cells that overexpressed the S-form (non-mitochondrial) of PHGPx (17Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). M15 cells, which overexpressed the L-form (mitochondrial) of PHGPx, were established by the transfection of RBL-2H3 cells with pSRα-L-form PHGPx and pSV2neo by electroporation, as described previously (17Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). A suspension of RBL-2H3 cells (1 × 107 cells/0.25 ml) was transferred to an electroporation cuvette (0.4-cm gap; Bio-Rad) with a total of 20 μg of linearized DNA, which consisted of 18 μg of each expression vector and 2 μg of pSV2neo, used to confer resistance to G418 (Geneticin; Life Technologies, Inc.) (21Southen P.J. Berg P. J. Mol. Appl. Genet. 1982; 1: 327-341PubMed Google Scholar). A potential difference of 250 V at 500 microfarads was applied at room temperature with a Gene Pulser II (Bio-Rad), and cell culture was reinitiated after a 10-min recovery period. Selection for resistance G418 (1 mg/ml) was initiated after 24 h, and cells were subsequently exposed to G418 at 0.5 mg/ml for 2 weeks. Individual G418-resistant colonies were isolated with cloning cylinders. Levels of expression of PHGPx were determined by immunoprecipitation with antibodies against PHGPx, and cells that overexpressed the L-form of PHGPx were isolated. Control cells and cells that overexpressed the L-form or the S-form of PHGPx were cultured in Dulbecco's modified Eagle's medium that contained 5% fetal calf serum and 0.5 mg/ml G418. Cells were labeled with 140 nCi/ml [75Se]sodium selenite (3126 Ci/g; MURR) for 96 h to determine the distribution of PHGPx and cGPx in cells. Confluent cells in 225-cm2 culture flasks were washed three times with phosphate-buffered saline (PBS) and harvested by treatment with trypsin. The cell suspension was centrifuged at 700 ×g for 5 min at room temperature and then [75Se]sodium selenite-labeled cells were fractionated as described previously (18Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The cell pellet was suspended in sucrose buffer (0.25 m sucrose, 1 mm EDTA, 3 mm imidazole, and 0.1% (v/v) ethanol, with leupeptin, antipain, chymostatin, and pepstatin A added at a final concentration of 10 μg/ml each and phenylmethylsulfonyl fluoride at a final concentration of 100 μg/ml, pH 7.2) and centrifuged at 700 ×g for 10 min at 4 °C. Pelleted cells were resuspended in the same buffer at approximately 1.5 × 107 cells/ml and homogenized with a Teflon/glass Potter-Elvehjem homogenizer. A nuclear fraction (pellet) and a postnuclear fraction (supernatant) were prepared by centrifugation at 700 × g for 10 min. The nuclear fraction was suspended in 200 μl of the sucrose buffer. Mitochondrial, microsomal, and cytosolic fractions from the postnuclear fraction were obtained by differential centrifugation as described by de Duve et al. (22de Duve C. Pressman B.C. Gianetto R. Wattiaux R. Appelmans F. Biochem. J. 1995; 60: 604-617Crossref Scopus (2573) Google Scholar). Each subcellular fraction was examined by standard enzymatic assays for activities of cytochrome coxidase (a mitochondrial marker), NADPH-cytochrome creductase (a microsomal marker), and lactate dehydrogenase (a cytosolic marker) as reported previously (23Roderick A.C. Michael F.M. Jan-Willem T. Methods Enzymol. 1995; 260: 117-132Crossref PubMed Scopus (125) Google Scholar, 24Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The distribution of histone H1, as a nuclear marker, was determined by immunoblotting with polyclonal antibodies against histone H1 (25Lawrence J.J. Daure M. Biochemistry. 1976; 15: 3301-3307Crossref PubMed Scopus (57) Google Scholar). The purity of each subcellular fraction of M15 cells was determined according to our previous paper in which the the purity of each organelle of control cells and non-mitochondrial PHGPx overexpressing cells including S1 and L9 cells has been determined (18Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The purity of each subfractionation in M15 cells was the same as those in S1 and L9 cells. Cytochromec oxidase was distributed in nuclear (7.9%),mitochondrial (75.6%), microsomal (3.5%), and cytosolic (12.9%) fractions of M15 cells. NADPH-cytochrome c reductase activities were found in nuclear (5.9%), mitochondrial (16.7%), microsomal (68.3%), and cytosolic (9.1%) fractions of M15 cells. The nuclear, mitochondrial, and microsomal fractions were solublized in 200 μl of 0.4% Triton X-100 in PBS for 2 h at 4 °C, and each solution was centrifuged at 100,000 × g for 1 h at 4 °C. The supernatants were supplemented into 400 μl each of PBS and subjected to immunoprecipitation with antibodies against PHGPx and cGPx, as described previously (17Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis (12.5% polyacrylamide) under non-reducing conditions. Gels were stained, dried, and subjected to autoradiography. Total levels of PHGPx and cGPx were calculated from results of scanning densitometry after autoradiography with a Bio-Imaging Analyzer (BAS2000; Fuji Film, Tokyo). Activities of PHGPx and cGPx were measured after the fractionation of cytosol and mitochondria from each cell line (1.5 × 108 cells). Mitochondrial fraction was sonicated and centrifuged at 10,000 × g for 10 min at 4 °C. The supernatants obtained from mitochondrial fraction and cytosolic fraction were used for assays of PHGPx and cGPx activities. PHGPx activity was determined by using phosphatidylcholine hydroperoxide (PCOOH) as the substrate according to the previous paper (18Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Activity of cGPx was determined by using hydrogen peroxide as the substrate (18Imai H. Narashima K. Arai M. Sakamoto H. Chiba N. Nakagawa Y. J. Biol. Chem. 1998; 273: 1990-1997Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The total activity of SOD was measured in terms of the percentage inhibition of the formation of superoxide by the xanthine-xanthine oxidase system (26Crapo J.D. Mccord J.M. Fridovich I. Methods Enzymol. 1978; 53: 382-393Crossref PubMed Scopus (619) Google Scholar). Mn-SOD activity was measured in the presence of 5 mm KCN, and Cu,Zn-SOD activity was calculated by subtraction of the activity of Mn-SOD from the total SOD activity. RBL-2H3 cells were transfected with plasmids that encoded GFP, L-GFP, or S-GFP by electroporation, as described above. Transfected cells were cultured on coverslips in 35-mm dishes in 2 ml of Dulbecco's modified Eagle's medium that contained 5% fetal calf serum at 37 °C in an atmosphere of 5% CO2 in air. After 24 h, cells were fixed for 20 min on coverslips with 4% formaldehyde and washed with Hanks' balanced salts solution. The fluorescence of cells was monitored and photographed with an Axiovert 135M inverted microscope (Carl Zwiss, Germany) equipped with a Planapochromat 63 × objective and a filter pack appropriate for GFP fluorescence. GFP and mitochondria were simultaneously detected in the same cells by the double staining with GFP fluorescence and a monoclonal antibody of Cy3-conjugated anti-cytochrome c oxidase subunit IV that was a specific probe for the mitochondrial staining (27Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (523) Google Scholar). Fixed cells were washed with phosphate-buffered saline (PBS) and were blocked with PBS containing 2% BSA at 25 °C for 30 min. The cells were incubated with 2 μg/ml mouse anti-cytochrome c oxidase subunit IV monoclonal antibodies diluted with 2% BSA-PBS at 25 °C for 2 h. Then the cells were washed with PBS and incubated with Cy3-conjugated goat anti-mouse IgG (Amersham Pharmacia Biotech) diluted to 10 μg/ml with PBS containing 2% BSA at 25 °C for 1 h. Fluorescence of GFP and Cy3 in the same cells was monitored and photographed with an appropriate filter pack. S1, L9, and M15 cells were plated at 0.5 × 105 cells/well in flat-bottomed 96-well culture plates and cultured for 24 h. Individual transformants were exposed to indicated doses of KCN, rotenone, CCCP, oligomycin, ort-BuOOH for appropriate periods. The LDH release assay was used for the determination of the cell viability, as described elsewhere (17Imai H. Sumi D. Sakamoto H. Hanamoto A. Arai M. Chiba N. Nakagawa Y. Biochem. Biophys. Res. Commun. 1996; 222: 432-438Crossref PubMed Scopus (69) Google Scholar). In one series of experiments, cells were incubated for 12 h prior to exposure to KCN with 0.5 mm buthionine sulfoxamine (BSO) for depletion of GSH. Changes in the integrity of plasma membrane and in the mitochondrial membrane potential were examined by monitoring staining with propidium iodide (PI) and Rh123, respectively. After treatment with KCN, cells were stained with PI (5 mg/ml) and Rh123 (1 mg/ml) for 10 min. We also used an oxidation-sensitive fluorescent probe, 5,6-carboxy-2′,7′-dichlorofluorescein-diacetate (DCFH-DA), to assess levels of intracellular peroxides, as follows. Cells were washed with PBS and incubated with 2.5 μm DCFH-DA in PBS for 15 min. DCFH-loaded cells were incubated with or without 25 mm KCN for the times indicated. The intensity fluorescence from PI, Rh123, and dichlorofluorescein (DCF) in cells was analyzed with a flow cytometer (EPICS® Elite Flow cytometer; Coulter, Hialeah, FL). Cellular levels of ATP were determined by the luciferin-luciferase method using a kit from Sigma (29Naderi S. Melchior D.L Anal. Biochem. 1990; 190: 304-308Crossref PubMed Scopus (6) Google Scholar). Cells (1 × 106 cells) were loaded with 20 mm cis-parinaric acid for 1.5 h at 37 °C and then washed with PBS. The loaded cells were treated with 25 mmKCN for the times indicated. After incubation, total lipids were extracted as described by Bligh and Dyer (30Bligh E.D. Dyer W. Can. J. Biochem. Physiol. 1959; 37: 911-918Crossref PubMed Scopus (43132) Google Scholar). Fluorescence of total lipids was monitored with a spectrofluorometric detector (RF-550; Shimazu Co. Ltd., Tokyo) with excitation at 303 nm and emission at 416 nm. Amounts of GSH in mitochondria and cytosol were measured according to the previous paper with a slight modification (28Imai K. Toyo'oka T. Watanabe Y. Anal. Biochem. 1983; 128: 471-473Crossref PubMed Scopus (162) Google Scholar). In brief, S1, L9, and M15 cells (each 2 × 107 cell) were fractionated into cytosol and mitochondria. Cytosol and mitochondria dispersed with the sonication were precipitated by the addition of trichloroacetic acid at a final concentration of 5%. After centrifugation at 10,000 × g for 10 min, GSH in the supernatant was converted to fluorescent derivative. The reaction was started by the addition of 0.5 ml of 0.02% ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) in 0.25m borate buffer, pH 10.5, which contained 5 mmEDTA and 1% tri-n-butyl phosphine. The reaction mixture kept at 60 °C for 30 min, and the reaction was terminated by the addition of 50 μl of 4 n HCl, and fluorescent thiol derivatives were separated by reversed-phase high pressure liquid chromatography (TSK-gel ODS; TOSOH Co. Ltd., Japan). The mobile phase was 0.1 m citrate buffer, pH 4.0, tetrahydrofuran/acetonitrile (94.8:0.2:5). The fluorescent thiol derivatives were monitored with emission at 516 nm and excitation at 384 nm during elution at a flow rate of 1.0 ml/min. Concentrations of protein were determined with the BCA protein assay reagent (Pierce), with bovine serum albumin (BSA) as the standard. All data from assays in which the number of replicates was three or more are expressed as mean values ± S.D. The L-form of PHGPx contains a leader sequence, but the S-form does not (Fig. 1). Chimeric proteins that included green fluorescent protein (GFP) were expressed in RBL-2H3 cells in order to determine whether the leader sequence of the L-form could serve to target GFP to the mitochondria of living cells. One fusion protein consisted of GFP with the leader sequence of 32 amino acids from the first residue of L-form (L-GFP) (Fig.1 B). The other was a fusion protein of GFP with 42 amino acids from the first residue of S-form (S-GFP) (Fig. 1 B). Expression vectors containing cDNA that encoded GFP,L-GFP, or S-GFP were used to transfect RBL-2H3 cells by electroporation and then the intracellular localization of fluorescence due to GFP was monitored with a fluorescence microscope 24 h later (Fig. 2). Fluorescence was diffusely distributed in cells that expressed S-GFP or GFP (Fig. 2, Aand B). By contrast, discrete regions with strong fluorescence were observed in cells that expressed L-GFP (Fig. 2 C). GFP and mitochondria in theL-GFP-transfected cells were simultaneously visualized by the double staining with GFP fluorescence and a monoclonal antibody of Cy3-conjugated anti-cytochrome c oxidase subunit IV (Fig. 2,C and D). The profile of fluorescence due to L-GFP was identical to that of mitochondrial cytochrome coxidase. Efficient import of GFP with the leader sequence of L-form into mitochondria indicates that the leader sequence at the amino terminus of mitochondrial PHGPx is the signal for targeting to mitochondria.Figure 2Targeting of L-GFP to the mitochondria of RBL-2H3 cells. Expression vectors encoding GFP (A), S-GFP (B), and L-GFP (C and D) were used to transfect RBL-2H3 cells. After the incubation for 24 h, fluorescent images of cells were photographed at higher magnification under a fluorescence microscope. RBL-2H3 cells that transfected with expression vector encoding L-GFP were double-stained with GFP fluorescence (C) and Cy3-conjugated anti-cytochromec oxidase monoclonal antibodies, which is a mitochondrion-specific probe (D). The black barhas a length of 10 μm.View Large Image Figure ViewerDownload (PPT) RBL-2H3 cells were transfected by electroporation with cDNAs that encoded theL-form and the S-form of PHGPx (Fig. 1 A). Two types of transformant that stably expressed substantial levels of PHGPx were isolated after appropriate selection. M15 cells strongly expressed the L-form of PHGPx with the leader sequence and L9 cells expressed the S-form of PHGPx. The control line of cells (S1) had been transfected with the expression vector without an insert. The three kinds of transformants were labeled with [75Se]sodium selenite for 4 days for determination of the amounts of PHGPx and cGPx (TableI). The total amounts of PHGPx in L9 and M15 cells were 4 and 3.5 times higher than that in S1 cells, respectively. No significant differences in total respective amounts of cGPx, Cu,Zn-SOD, and Mn-SOD were detected among L9, M15, and S1 cells.Table IThe levels of selenium-labeled PHGPx and cGPx and of Mn-SOD and Cu,Zn-SOD in S1, L9, and M15 cellsStrain75Se-PHGPx75Se-cGPxMn-SODCu,Zn-SODControl line of cellsS14.62 ± 0.13 × 10421.4 ± 0.8 × 1040.21 ± 0.029.66 ± 0.61Non-mitochondrial PHGPx-overexpressing cellsL918.0 ± 0.7 × 10420.6 ± 0.9 × 1040.18 ± 0.0310.70 ± 2.73Mitochondrial PHGPx-overexpressing cellsM1516.1 ± 0.6 × 10420.8 ± 0.8 × 1040.24 ± 0.0110.00 ± 1.52The amounts of PHGPx and cGPx were determined by measurements of
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