Phospholipid Hydroperoxides Are Substrates for Non-selenium Glutathione Peroxidase
1999; Elsevier BV; Volume: 274; Issue: 30 Linguagem: Inglês
10.1074/jbc.274.30.21326
ISSN1083-351X
AutoresAron B. Fisher, Chandra Dodia, Yefim Manevich, Jinwen Chen, Sheldon I. Feinstein,
Tópico(s)Sulfur Compounds in Biology
ResumoThis study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity withsn-2-linolenoyl- orsn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM−1min−1) were k 1 = 1–3 × 105 and k 2 = 4–11 × 104. In the presence of 0.36 mm GSH, apparentK m was 120–130 μm and apparentV max was 1.5–1.6 μmol/min/mg of protein. Assays with H2O2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7–8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 μm. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems. This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity withsn-2-linolenoyl- orsn-2-arachidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM−1min−1) were k 1 = 1–3 × 105 and k 2 = 4–11 × 104. In the presence of 0.36 mm GSH, apparentK m was 120–130 μm and apparentV max was 1.5–1.6 μmol/min/mg of protein. Assays with H2O2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7–8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 μm. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems. The integrity of biomembranes and other phospholipid-enriched cellular components requires a mechanism to repair oxidized phospholipids generated by spontaneous or pathologic oxidation. Intracellular glutathione peroxidase (GSH Px) 1The abbreviations used are: GSH Px, glutathione peroxidase; NSGPx, non-selenium glutathione peroxidase; hNSGPx, human NSGPx; PHGPx, phospholipid hydroperoxide glutathione peroxidase; PLA2, phospholipase A2; DTT, dithiothreitol; PLPC, 1-palmitoyl-2-linolenoyl-sn-glycerol-3-phosphocholine; PAPC, 1-palmitoyl-2-arachidonoyl-snglycerol-3-phosphocholine; DENP, diethyl p-nitrophenyl phosphate; MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol; pBPB, p-bromophenacyl bromide; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; MES, 4-morpholinoethanesulfonic acid plays an important role in reducing fatty acid hydroperoxides and H2O2, although this enzyme has no activity toward phospholipid hydroperoxides (1Michiels C. Raes M. Toussaint O. Remacle J. Free Radical Biol. Med. 1994; 17: 235-248Crossref PubMed Scopus (1044) Google Scholar). Detoxification of phospholipid hydroperoxides can be accomplished through the combined enzymatic activity of phospholipase A2(PLA2) and reduction of the resultant fatty acid hydroperoxides with GSH Px (2van Kuijk F.J.G.M. Sevanian A. Handelman G.J. Dratz E.A. Trends Biochem. Sci. 1987; 12: 31-34Abstract Full Text PDF Scopus (372) Google Scholar). Recently, a phospholipid hydroperoxide GSH Px (PHGPx) has been described as a 19-kDa selenoenzyme that uses GSH to reduce peroxidized phospholipids to the nontoxic hydroxy derivative (3Ursini F. Maiorino M. Gregolin C. Biochim. Biophys. Acta. 1985; 839: 62-70Crossref PubMed Scopus (765) Google Scholar, 4Maiorino M. Gregolin C. Ursini F. Methods Enzymol. 1990; 186: 448-457Crossref PubMed Scopus (227) Google Scholar, 5Brigelius-Flohé R. Aumann K-D. Blöcker H. Gross G. Kiess M. Klöppel K-D. Maiorino M. Roveri A. Schuckelt R. Ursini F. Wingender E. Flohé L. J. Biol. Chem. 1994; 269: 7342-7348Abstract Full Text PDF PubMed Google Scholar). Reduction of phospholipid hydroperoxides also has been described as a minor activity for the selenoenzyme plasma GSH Px and for some GSH S-transferase enzymes (6Yamamoto Y. Takahashi K. Arch. Biochem. Biophys. 1993; 305: 541-545Crossref PubMed Scopus (134) Google Scholar, 7Hurst R. Bao Y. Jemth P. Mannervik B. Williamson G. Biochem. J. 1998; 332: 97-100Crossref PubMed Scopus (128) Google Scholar). In this report, we investigated phospholipid hydroperoxides as substrates for a novel 25-kDa GSH Px. The protein was first isolated from the bovine ciliary body and was shown to catalyze the reduction of H2O2 and organic hydroperoxides using GSH as electron donor (8Shichi H. Demar J.C. Exp. Eye Res. 1990; 50: 513-520Crossref PubMed Scopus (57) Google Scholar). Activity toward phospholipid hydroperoxides was not tested. The absence of selenium in the protein was demonstrated by assay with 2,3-diaminonaphthalene (8Shichi H. Demar J.C. Exp. Eye Res. 1990; 50: 513-520Crossref PubMed Scopus (57) Google Scholar). The enzyme, which was called a non-selenium GSH Px (NSGPx), had no GSH S-transferase activity when assayed with a spectrum of potential SH acceptors (8Shichi H. Demar J.C. Exp. Eye Res. 1990; 50: 513-520Crossref PubMed Scopus (57) Google Scholar). A full-length cDNA sequence (HA0683) 2GenBankTM accession numberD14662. ultimately shown to encode NSGPx was first isolated from a human myeloblast cell line (KG-1) (9Nomura N. Miyajima N. Sazuka T. Tanaka A. Kawarabayasi Y. Sato S. Nagase T. Seki N. Ishikawa K. Tabata S. DNA Res. 1994; 1: 27-35Crossref PubMed Scopus (272) Google Scholar, 10Nagase T. Miyajima N. Tanaka A. Sazuka T. Seki N. Sato S. Tabata S. Ishikawa K. Kawarabayasi Y. Kotani H. Nomura N. DNA Res. 1995; 2: 37-43Crossref PubMed Scopus (114) Google Scholar, 11Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar) and described as a Ca2+-independent PLA2 (12Kim T-S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Subsequently, an identical cDNA from a human lymphoma cell line (U937) was described; protein encoded by this cDNA was expressed in E. coli and NIH 3T3 cells and shown to be a peroxidase with minimal PLA2 activity (13Kang S-W. Baines I.C. Rhee S-G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). In contrast to the prior publication using the native protein (8Shichi H. Demar J.C. Exp. Eye Res. 1990; 50: 513-520Crossref PubMed Scopus (57) Google Scholar), anin vitro assay with the protein product of this clone indicated that GSH was not an effective reductant, and the report concluded that the physiologic reductant for the peroxidase was unknown (13Kang S-W. Baines I.C. Rhee S-G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Based on homology to the peroxiredoxin family, the enzyme was called 1-Cys peroxiredoxin, since only one Cys residue was conserved (13Kang S-W. Baines I.C. Rhee S-G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). More recently, protein expressed in vitro with a bovine cDNA clone was found to utilize GSH as a reductant for peroxidase activity, although the discrepancy with the previous study was not explained (14Singh A.K. Shichi H. J. Biol. Chem. 1998; 273: 26171-26178Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). One possibility, although unlikely, is that the human and bovine enzymes have different cofactor requirements. The present study investigated substrate utilization of recombinant NSGPx using the human clone (HA0683) that has been previously described (12Kim T-S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) as well as a newly isolated cDNA clone from a bovine lung cDNA library. Our results with both human and bovine NSGPx show that GSH is effective for reduction of H2O2 and organic hydroperoxides including phospholipid hydroperoxides. GSH, glutathione reductase, β-NADPH, isopropyl β-d-thiogalactopyranoside, diethylp-nitrophenyl phosphate (DENP), p-bromophenacyl bromide (pBPB), dithiothreitol (DTT), H2O2, cumene hydroperoxide, t-butyl hydroperoxide, linolenic acid, arachidonic acid, 1-chloro-2,4-dinitrobenzene, GSH peroxidase (from bovine erythrocytes), GSH S-transferase (from rat liver), and soybean lipoxidase (EC 1.1311.12 type V) were purchased from Sigma. Phospholipids, 1-palmitoyl-2-arachidonoyl PC (PAPC) and 1-palmitoyl-2-linolenoyl PC (PLPC), were purchased from Avanti Polar Lipids (Alabaster, AL). 13-Hydroperoxyoctadecanoic acid and 15-hydroperoxyeicosatetranoic acid were purchased from Cayman Chemicals (Ann Arbor, MI). The C18 Sep-Pack was from Millipore Corp. (Milford, MA). Radiochemicals were purchased from DuPont NEN. Molecular mass standards for SDS-PAGE, transblot membrane, protein dye-binding assay kit, horseradish peroxidase-conjugated goat anti-rabbit IgG, and Triton X-100 (electrophoretically purified) were purchased from Bio-Rad. Enhanced chemiluminescence kit and x-ray film were purchased from Amersham Pharmacia Biotech. pET28C vector and the His bind kit containing imidazole buffer were purchased from Novagen (Madison, WI). A bovine lung cDNA library was purchased from Stratagene (La Jolla, CA). Lennox LB broth was obtained from Fisher. 1-Hexadacyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), a PLA2 inhibitor, was a gift from Dr. Mahendra Jain (University of Delaware). The human cDNA clone (HA0683) (12Kim T-S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) was digested with the restriction enzyme HindIII, and the insert was recloned into theHindIII site of pET28C (Novagen, Madison, WI) in both the forward (sense) and reverse (antisense) directions. The inserted fragment contained about 1040 nucleotide pairs and included the entire coding sequence of the human enzyme along with 43 nucleotides from the 5′-untranslated region and 320 nucleotides from the 3′-untranslated region. Plasmid DNAs were prepared using a QIAGEN column (QIAGEN, Chatsworth, CA); were transformed into E. coli BL21(DE3) cells, which produce T7 RNA polymerase and express pET28C inserts efficiently; and were incubated at 37 °C overnight to get colonies. A single colony was inoculated in 50 ml of LB broth containing kanamycin (30 μg/ml) and incubated with shaking at 37 °C until optical density at 600 nm reached 0.6. Recombinant protein expression was induced by adding isopropyl β-d-thiogalactopyranoside to 1 mm final concentration and incubating at 37 °C for 3 h. The cells were harvested by centrifugation at 5000 ×g for 5 min at 4 °C, resuspended in 12.5 ml of cold 50 mm Tris-HCl (pH 8.0), and recentrifuged. The cell pellet was resuspended in 4 ml of cold 50 mm Tris-HCl and sonicated three times. For purification, the cell pellet was resuspended and sonicated in 5 mm imidazole buffer. The HA0683 product was expressed as a fusion protein with a series of six histidine residues in pET28C. This "His tag" allowed affinity purification of the fusion protein on a Ni2+ column using the His bind kit under native conditions by following the protocols recommended by the manufacturer. Briefly, after allowing the 5 mm imidazole buffer to drain to the top of the column bed (2.5 ml), the column was loaded with the prepared extract in the same buffer. The flow rate was 10 ml/h, the column was washed with 25 ml of 5 mm imidazole buffer and then washed with 15 ml 60 mm imidazole buffer, and the bound protein was eluted with 15 ml of 1 m imidazole buffer. Fractions of 1 ml were collected from the column and analyzed by SDS-PAGE (15Kim T-S. Dodia C. Chen X. Hennigan B.B. Jain M. Feinstein S.I. Fisher A.B. Am. J. Physiol. 1998; 274: L750-L761PubMed Google Scholar). NSGPx as indicated by Western blot was concentrated in fractions 2–5, which were pooled. Western blot of the purified protein utilized a previously described polyclonal antibody prepared in rabbits to a 15-amino acid synthetic peptide representing a conserved internal sequence of NSGPx or a mouse monoclonal antibody (8H11) raised against the E. coli-expressed HA0683 product (human NSGPx) (15Kim T-S. Dodia C. Chen X. Hennigan B.B. Jain M. Feinstein S.I. Fisher A.B. Am. J. Physiol. 1998; 274: L750-L761PubMed Google Scholar). HA0683 also was expressed with a wheat germ expression system in the presence of 0.5 mm [35S]methionine (63 Ci/mmol) as described previously; this procedure generated a single band of 35S-labeled protein by autoradiography of SDS-PAGE gels (12Kim T-S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Each translation included a negative control in which no RNA was added. An aliquot of the translated proteins was separated by SDS-PAGE, and the radiolabeled protein band was cut from the gel and analyzed by scintillation counting. The amount of radiolabeled NSGPx representing the translated cDNA product was calculated from total incorporated 35S, [35S]methionine specific activity of the starting material, and the fractional methionine content of NSGPx as deduced from the cDNA. After translation, NSGPx represented 0.05% of the total wheat germ extract protein. A similar protocol, but with nonlabeled methionine, was used to express bovine NSGPx using the bovine lung cDNA. The hydroperoxides of linolenic acid, arachidonic acid, PLPC, and PAPC were prepared by treatment of the corresponding fatty acid or phospholipid with soybean 15-lipoxidase according to previously described methods (4Maiorino M. Gregolin C. Ursini F. Methods Enzymol. 1990; 186: 448-457Crossref PubMed Scopus (227) Google Scholar). The reactions were monitored by continuous recording of oxygen consumption using a standard oxygen electrode. The hydroperoxides of linolenic and arachidonic acids, 13-hydroperoxyoctadecanoic acid and 15-hydroperoxyeicosatetranoic acid, respectively, were identified by authentic standards and separated by reverse phase HPLC in a mobile phase consisting of acetonitrile/water/acetic acid (60:40:0.1) or methanol/water/acetic acid (85:15:0.1) with UV detection at 235 nm (16Funk C.D. Gunne H. Steiner H. Izumi T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2592-2596Crossref PubMed Scopus (41) Google Scholar). The PLPC and PAPC hydroperoxides were separated by reverse phase HPLC in a mobile phase consisting of 10 mm ammonium acetate (pH 5.0) and methanol (1:20, v/v) (17Therod P. Couturier M. Demelier T.F. Lemonnier F. Lipids. 1993; 28: 245-249Crossref PubMed Scopus (45) Google Scholar) with a flow rate of 1 ml/min on a Hewlett-Packard Series 1050 system equipped with a Columbus 5-μm C18 column (250 × 4.6 mm) (Phenomenex, Torrance, CA). Absorbance was measured at 205 nm to detect the phospholipid and at 235 nm to detect conjugated dienes. Peak retention time was 28.6 min for the starting phospholipid, 23.4 min for the hydroxyphospholipid, and 20.9 min for the hydroperoxide, with an additional peak coming earlier (17.3 min) with oxidized PAPC, suggesting the presence of a double hydroperoxidation product. The products were recovered between 20 and 23 min, and the hydroperoxide concentrations were estimated from the integrated peaks based on an extinction coefficient of 2.3 × 104 cm−1m−1 (18Gardner H.W. Biochim. Biophys. Acta. 1989; 1001: 274-281Crossref PubMed Scopus (176) Google Scholar). The HPLC-purified lipid was used as substrate for enzymatic assays. NSGPx activity was assayed by measuring consumption of NADPH in the presence of GSH and GSH reductase. Unless otherwise noted, all assays used the HA0683 product expressed inE. coli and affinity-purified. The standard reaction buffer (3 ml) was 50 mm Tris-HCl, 2 mmNaN3, 0.1 mm EDTA (pH 8.0), 0.3 mmNADPH, 0.36 mm GSH, and 0.23 units/ml GSH reductase. To assay PC hydroperoxides, 0.1% Triton X-100 was routinely added, since activity was approximately 40% less in its absence. For the standard assay, the mixture plus enzyme (2 μg of protein/ml) was preincubated for 5 min with continuous stirring. Fluorescence was continuously recorded at 460 nm (340-nm excitation) using a fluorescence spectrophotometer (Photon Technology Instruments, Bricktown, NJ). After a steady base line was achieved, the reaction was started by the addition of substrate generally at 250 μm, and the linear change in fluorescence was recorded for 5–10 min. The change in fluorescence was corrected for the relatively small base-line nonenzymatic oxidation of NADPH and used to calculate enzyme activity based on authentic NADPH standards. Protein was measured with Coomassie Blue (Bio-Rad protein dye binding kit) using bovine γ-globulin as a standard. Enzymatic activity was expressed as nmol of NADPH oxidized/min/mg of protein. Data for multiple replications are presented as mean ± S.E. To confirm results with the fluorescence assay, the production of hydroxy-PLPC from PLPC hydroperoxide was determined by HPLC analysis, assuming that the extinction coefficient at 235 nm is the same for both molecular species (18Gardner H.W. Biochim. Biophys. Acta. 1989; 1001: 274-281Crossref PubMed Scopus (176) Google Scholar). In some assays, NSGPx activity was determined by the disappearance of H2O2 by modification of the method of Kanget al. (13Kang S-W. Baines I.C. Rhee S-G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). The enzyme (6 μg/ml) was incubated for 2 or 10 min in 50 mm Tris, 0.1 mm EDTA buffer with 0.2 or 5 mm GSH and 250 μmH2O2. At the end of incubation, 2 mmFe(NH4)2(SO4)2 and 0.25 mm KSCN were added. The absorbance of the red ferrithiocyanate complex formed in the presence of peroxides was measured at 480 nm. This assay was used to test pH dependence; buffers were 50 mm glycine (pH 3), 50 mm sodium acetate (pH 4–5), 50 mm MES (pH 6), or 50 mm Tris-HCl (pH 7–10). This assay also was used to measure peroxidase activity in the presence of 2 mm DTT, substituting for GSH, as described previously (13Kang S-W. Baines I.C. Rhee S-G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). GSH S-transferase activity was measured by continuous spectrophotometric assay with 1-chloro-2,4-dinitrobenzene substrate (8Shichi H. Demar J.C. Exp. Eye Res. 1990; 50: 513-520Crossref PubMed Scopus (57) Google Scholar). Since the initial isolation of this enzyme was from bovine sources (8Shichi H. Demar J.C. Exp. Eye Res. 1990; 50: 513-520Crossref PubMed Scopus (57) Google Scholar), we isolated a bovine cDNA to complement previous descriptions of human, rat, and mouse clones (12Kim T-S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 15Kim T-S. Dodia C. Chen X. Hennigan B.B. Jain M. Feinstein S.I. Fisher A.B. Am. J. Physiol. 1998; 274: L750-L761PubMed Google Scholar, 19Frank S. Munz B. Werner S. Oncogene. 1997; 14: 915-921Crossref PubMed Scopus (109) Google Scholar, 20Munz B. Frank S. Hübner G. Olsen E. Werner S. Biochem. J. 1997; 326: 579-585Crossref PubMed Scopus (106) Google Scholar). The cDNA was isolated and sequenced prior to the recent report of a bovine clone for this enzyme isolated by polymerase chain reaction (14Singh A.K. Shichi H. J. Biol. Chem. 1998; 273: 26171-26178Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). We screened a commercially available bovine lung cDNA library cloned in bacteriophage λ Uni-ZAP XR. Approximately 50,000 plaques were screened using a 589-nucleotide polymerase chain reaction product derived from HA0683 as a probe. Forty positive clones were obtained, representing 0.08% of those screened. Selected positive bovine clones were "zapped" into Bluescript plasmid according to the supplier's instructions and sequenced in cooperation with the DNA Sequencing Facility at the University of Pennsylvania. The E. coli expression system using the sense orientation for the human cDNA (HA0683) generated a protein (called hNSGPx) with an apparent molecular mass of 33 kDa on SDS-PAGE and Western blot probed with the polyclonal NSGPx anti-peptide antibody (Fig. 1). This protein band was not visible when the antisense orientation was used (not shown). A similar result for Western blot was obtained when probed with a monoclonal antibody to the human fusion protein (hNSGPx) (not shown). The larger apparent mass for the fusion protein on PAGE compared with the mass of 25,032 Da deduced from HA0683 (12Kim T-S. Sundaresh C.S. Feinstein S.I. Dodia C. Skach W.R. Jain M. Nagase T. Seki N. Ishikawa K. Nomura N. Fisher A.B. J. Biol. Chem. 1997; 272: 2542-2550Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) is due in part to the presence of the "His tag" plus additional amino acids from the vector and from the 5′ normally untranslated region; we estimate the mass of these additional amino acids as 5996 Da. Based on densitometric scanning of Coomassie Blue-stained gels, the expressed hNSGPx protein in the crude cell homogenate was 13% of total protein (not shown), and this increased to 85% after the Ni2+ column (Fig. 1). This figure for purity represents a lower limit, since immunoreactivity at higher molecular mass on the Western blot suggests the presence of dimers or multimeric forms. The expressed hNSGPx demonstrated GSH peroxidase activity as indicated by the linear decrease in NADPH fluorescence following the addition of organic hydroperoxide substrate to the assay buffer (Fig.2). Fig. 2 A showst-butyl hydroperoxide as substrate; H2O2 and hydroperoxides of cumene, linolenic acid, and arachidonic acid were equally effective (TableI). There was zero activity (not shown) with the antisense preparation, indicating specificity of the translated gene fragment. Hydroperoxides of phosphatidylcholine likewise were reduced by hNSGPx (Fig. 2 B). PLPC and PAPC hydroperoxides were equally effective substrates (Table I). Enzymatic activity required GSH, which in combination with glutathione reductase resulted in oxidation of NADPH (Fig. 2 C). NADPH itself was not a cofactor for the enzyme, and substitution of thioredoxin/thioredoxin reductase for GSH/glutathione reductase also was ineffective (Fig. 2 D). In contrast to hNSGPx, authentic GSH Px (selenoenzyme from erythrocytes) reduced H2O2 and t-butyl hydroperoxide in the presence of GSH but had no reactivity toward PLPC hydroperoxide (Fig. 3).Table INSGPx activity with various hydroperoxide (HP) substratesSubstrateNSGPx activityV max(app)K m(app)k 1k 2nmol/min/mg proteinnmol/min/mg proteinμmmm−1min−1mm−1 min−1H2O21850 ± 3218101801.8 × 1059.0 × 104Cumene HP1140 ± 3011201201.2 × 1054.0 × 104t-Butyl HP1320 ± 3912701421.6 × 1056.1 × 104Linolenoyl HP1400 ± 2113901411.7 × 1056.8 × 104Arachidonoyl HP1360 ± 3013801351.8 × 1057.2 × 104PLPC HP1470 ± 3715001203.2 × 10511 × 104PAPC HP1670 ± 2916401292.3 × 1057.9 × 104Activity for hNSGPx was measured with the standard assay (see Fig. 2) at 250 μm substrate concentration and 0.36 mmGSH. Values represent mean ±S.E. for n = 3 separate experiments. Apparent kinetic constantsV max and K m were calculated from double-reciprocal plots of activity versus varying substrate concentration at 0.36 mm GSH as illustrated in Fig.6 A. The kinetic constants k 1 andk 2 were calculated from double-reciprocal plots of activity versus varying substrate concentration at different fixed concentrations of GSH as shown in Fig. 6 C and described under "Results." Since the peroxidase reaction does not saturate with GSH, the K m is indefinite; an apparent K m for a given [GSH] can be calculated from the relationship [GSH]k 2/k 1. Open table in a new tab Figure 3Same conditions as in Fig. 2 using bovine erythrocyte GSH Px (0.1 unit/ml) as enzyme instead of NSGPx. This enzyme can reduce t-butyl hydroperoxide (t-BUOOH) and H2O2 but not PLPC hydroperoxide (PLPCOOH).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Activity for hNSGPx was measured with the standard assay (see Fig. 2) at 250 μm substrate concentration and 0.36 mmGSH. Values represent mean ±S.E. for n = 3 separate experiments. Apparent kinetic constantsV max and K m were calculated from double-reciprocal plots of activity versus varying substrate concentration at 0.36 mm GSH as illustrated in Fig.6 A. The kinetic constants k 1 andk 2 were calculated from double-reciprocal plots of activity versus varying substrate concentration at different fixed concentrations of GSH as shown in Fig. 6 C and described under "Results." Since the peroxidase reaction does not saturate with GSH, the K m is indefinite; an apparent K m for a given [GSH] can be calculated from the relationship [GSH]k 2/k 1. hNSGPx also reduced H2O2 with DTT as electron donor as determined with the end point assay, consistent with previous reports (13Kang S-W. Baines I.C. Rhee S-G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 23Peshenko I.V. Novoselov V.I. Evdokimov V.A. Nikolaev Y.V. Kamzalov S.S. Shuvaeva T.M. Lipkin V.M. Fesenko E.E. Free Radical Biol. Med. 1998; 25: 654-659Crossref PubMed Scopus (48) Google Scholar). Peroxidase activity of the crude cell lysate fromE. coli prior to hNSGPx affinity purification was 61 ± 2 nmol/min/mg of protein (n = 4) for the end point assay, and no activity was seen in the absence of DTT. The corresponding activity in nmol/min/mg of protein with this preparation using 5 mm GSH in place of DTT was 60 ± 1 (n = 4). Activity for affinity-purified hNSGPx assayed with 0.2 mm GSH was 1275 ± 16 nmol/min/mg of protein (n = 4). The effect of pH on the peroxidase reaction was evaluated with affinity-purified hNSGPx by the disappearance of H2O2 using the end point assay with 0.2 mm GSH. The pH optimum was in the range of 7–8; activity was reduced by 65% at pH 6, and virtually no activity was seen at pH 5 and below (Fig. 4). The pH dependence for PLPC hydroperoxide reduction was evaluated at pH 5 and 8 using HPLC to determine formation of the hydroxyphospholipid. HPLC of the purified original substrate showed a phospholipid hydroperoxide peak at 20.9 min but no hydroxyphospholipid peak. After a 1-h incubation of hNSGPx with substrate at pH 8, the peak height of the hydroperoxide signal was significantly decreased, and a hydroxyphospholipid peak appeared at 23.4 min (Fig. 5 C); hNSGPx activity calculated from the integrated hydroxyphospholipid peak was 1400 nmol/min/mg of protein, similar to the value obtained with the fluorescence assay. No hydroxyphospholipid peak was detected at pH 5, indicating the absence of NSGPx activity (Fig. 5 D).Figure 5pH dependence of NSGPx activity with PLPC hydroperoxide substrate analyzed by HPLC. hNSGPx (2 μg/ml) was incubated for 1 h with 250 μm PLPC hydroperoxide and 0.36 mm GSH in 50 mm Tris-HCl (pH 8) or 50 mm sodium acetate (pH 5) buffer. The reaction was stopped by passage through a C18 Sep-Pack and elution of the final product with methanol. Samples were concentrated 5-fold with a Speed Vac and then analyzed by reversed phase HPLC. The elution time is indicated in minutes. The peak at 20.9 min indicates the PLPC hydroperoxide, while the hydroxyphospholipid peak was at 23.4 min. Absorbance of PLPC hydroperoxide substrate before incubation was measured at 205 nm (A) to demonstrate phospholipid and at 235 nm (B) to show conjugated dienes. C, incubation at pH 8 resulted in a decrease in the PLPC hydroperoxide peak and increase in hydroxy-PLPC peak indicating reduction of the oxidized PLPC substrate. D, with incubation at pH 5, there was no change in the PLPC hydroperoxide and hydroxy-PLPC peaks, indicating no NSGPx activity at this pH.View Large
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