Title: Peroxynitrite Irreversibly Inactivates the Human Xenobioticmetabolizing Enzyme Arylamine N-Acetyltransferase 1 (NAT1) in Human Breast Cancer Cells
Abstract: Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (kinact = 5 × 104m–1·s–1) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1. Arylamine N-acetyltransferases (NATs) play an important role in the detoxification and metabolic activation of a variety of aromatic xenobiotics, including numerous carcinogens. Both of the human isoforms, NAT1 and NAT2, display interindividual variations, and associations between NAT genotypes and cancer risk have been established. Contrary to NAT2, NAT1 has a ubiquitous tissue distribution and has been shown to be expressed in cancer cells. Given that the activity of NAT1 depends on a reactive cysteine that can be a target for oxidants, we studied whether peroxynitrite, a highly reactive nitrogen species involved in human carcinogenesis, could inhibit the activity of endogenous NAT1 in MCF7 breast cancer cells. We show here that exposure of MCF7 cells to physiological concentrations of peroxynitrite and to a peroxynitrite generator (3-morpholinosydnonimine N-ethylcarbamide, or SIN1) leads to the irreversible inactivation of NAT1 in cells. Further kinetic and mechanistic analyses using recombinant NAT1 showed that the enzyme is rapidly (kinact = 5 × 104m–1·s–1) and irreversibly inactivated by peroxynitrite. This inactivation is due to oxidative modification of the catalytic cysteine. We conclude that the reducing cellular environment of MCF7 cells does not sufficiently protect NAT1 from peroxynitrite-dependent inactivation and that only high concentrations of reduced glutathione could significantly protect NAT1. Thus, cellular generation of peroxynitrite may contribute to carcinogenesis and tumor progression by weakening key cellular defense enzymes such as NAT1. Acetylation is a major biotransformation pathway for various aromatic amines, including drugs and carcinogens. Many arylamines, such as 4-aminobiphenyl (a tobacco-associated compound) and benzidine (occupational exposure) are important environmental carcinogens (1Badawi A.F. Hirvonen A. Bell D.A. Lang N.P. Kadlubar F.F. Cancer Res. 1995; 55: 5230-5237PubMed Google Scholar, 2Hein D.W. McQueen C.A. Grant D.M. Goodfellow G.H. Kadlubar F.F. Weber W.W. Drug Metab. Dispos. 2000; 28: 1425-1432PubMed Google Scholar, 3Hein D.W. Mutat. Res. 2002; 506-507: 65-77Crossref PubMed Scopus (424) Google Scholar), and their biotransformation through N- and/or O-acetylation has been linked to carcinogenesis (2Hein D.W. McQueen C.A. Grant D.M. Goodfellow G.H. Kadlubar F.F. Weber W.W. Drug Metab. Dispos. 2000; 28: 1425-1432PubMed Google Scholar, 3Hein D.W. Mutat. Res. 2002; 506-507: 65-77Crossref PubMed Scopus (424) Google Scholar, 4Boelsterli U.A. Mechanistic Toxicology: The Molecular Basis of How Chemicals Disrupt Biological Targets. Taylor and Francis Group, London and New York2003Crossref Google Scholar, 5Butcher N.J. Boukouvala S. Sim E. Minchin R.F. Pharmacogenomics J. 2002; 2: 30-42Crossref PubMed Scopus (152) Google Scholar). In humans, these reactions are catalyzed by two xenobiotic-metabolizing enzymes (XME), 1The abbreviations used are: XME, xenobiotic-metabolizing enzymes; NAT, arylamine N-acetyltransferase; SIN1, 3-morpholinosydnonimine N-ethylcarbamide; PAS, p-aminosalicylic acid; PNPA, p-nitrophenylacetate; AcCoA, acetyl-coenzyme A; DTT, 1,4-dithiothreitol; PBS, phosphate-buffered saline. arylamine N-acetyltransferase 1 and arylamine N-acetyltransferase 2 (NAT1 and NAT2; EC 2.3.1.5). Both of the human isoforms, NAT1 and NAT2, display interindividual variations and associations between NAT genotypes, and cancer risk has been established (1Badawi A.F. Hirvonen A. Bell D.A. Lang N.P. Kadlubar F.F. Cancer Res. 1995; 55: 5230-5237PubMed Google Scholar, 3Hein D.W. Mutat. Res. 2002; 506-507: 65-77Crossref PubMed Scopus (424) Google Scholar, 5Butcher N.J. Boukouvala S. Sim E. Minchin R.F. Pharmacogenomics J. 2002; 2: 30-42Crossref PubMed Scopus (152) Google Scholar). In addition, these cytosolic enzymes are encoded by two separate genes located on 8p22, a chromosomal region commonly deleted in certain human cancers (5Butcher N.J. Boukouvala S. Sim E. Minchin R.F. Pharmacogenomics J. 2002; 2: 30-42Crossref PubMed Scopus (152) Google Scholar, 6Matas N. Thygesen P. Stacey M. Risch A. Sim E. Cytogenet. Cell Genet. 1997; 77: 290-295Crossref PubMed Scopus (86) Google Scholar, 7Blum M. Grant D.M. McBride W. Heim M. Meyer U.A. DNA Cell Biol. 1990; 9: 193-203Crossref PubMed Scopus (468) Google Scholar). This raised the possibility that the absence of NATs or their inactivation may contribute to carcinogenesis and/or tumor progression (6Matas N. Thygesen P. Stacey M. Risch A. Sim E. Cytogenet. Cell Genet. 1997; 77: 290-295Crossref PubMed Scopus (86) Google Scholar, 8Butcher N.J. Ilett K.F. Minchin R.F. Mol. Pharmacol. 2000; 57: 468-473Crossref PubMed Scopus (80) Google Scholar, 9Butcher N.J. Ilett K.F. Minchin R.F. Biochem. Pharmacol. 2000; 60: 1829-1836Crossref PubMed Scopus (29) Google Scholar). Despite their high degrees of sequence identity, NAT1 and NAT2 differ markedly in terms of amine-containing acceptor substrates (10Grant D.M. Blum M. Beer M. Meyer U.A. Mol. Pharmacol. 1991; 39: 184-191PubMed Google Scholar, 11Pompeo F. Brooke E. Kawamura A. Mushtaq A. Sim E. Pharmacogenomics. 2002; 3: 19-30Crossref PubMed Scopus (61) Google Scholar) and tissue distribution (2Hein D.W. McQueen C.A. Grant D.M. Goodfellow G.H. Kadlubar F.F. Weber W.W. Drug Metab. Dispos. 2000; 28: 1425-1432PubMed Google Scholar, 12Sim E. Payton M. Noble M. Minchin R. Hum. Mol. Genet. 2000; 9: 2435-2441Crossref PubMed Scopus (85) Google Scholar). Indeed, NAT2 is primarily located in the liver and colon epithelium (13Deguchi T. J. Biol. Chem. 1992; 267: 18140-18147Abstract Full Text PDF PubMed Google Scholar, 14Ilett K.F. Ingram D.M. Carpenter D.S. Teitel C.H. Lang N.P. Kadlubar F.F. Minchin R.F. Biochem. Pharmacol. 1994; 47: 914-917Crossref PubMed Scopus (68) Google Scholar), whereas NAT1 seems ubiquitous (15Meisel P. Pharmacogenomics. 2002; 3: 349-366Crossref PubMed Scopus (52) Google Scholar, 16Rodrigues-Lima F. Cooper R.N. Goudeau B. Atmane N. Chamagne A.M. Butler-Browne G. Sim E. Vicart P. Dupret J.M. J. Histochem. Cytochem. 2003; 51: 789-796Crossref PubMed Scopus (35) Google Scholar). In addition, NAT1 expression has been demonstrated in different cancers and especially in human breast cancer, where it may play a role in cancer progression (17Adam P.J. Berry J. Loader J.A. Tyson K.L. Craggs G. Smith P. De Belin J. Steers G. Pezzella F. Sachsenmeir K.F. Stamps A.C. Herath A. Sim E. O'Hare M.J. Harris A.L. Terrett J.A. Mol. Cancer Res. 2003; 1: 826-835PubMed Google Scholar). NAT isoforms have been detected in a number of species, from bacteria to mammals (11Pompeo F. Brooke E. Kawamura A. Mushtaq A. Sim E. Pharmacogenomics. 2002; 3: 19-30Crossref PubMed Scopus (61) Google Scholar, 18Rodrigues-Lima F. Blomeke B. Sim E. Dupret J.M. Pharmacogenomics J. 2002; 2: 152-155Crossref PubMed Scopus (11) Google Scholar). Crystallographic determination of the structure of the NATs from Salmonella typhimurium and Mycobacterium smegmatis, and the subsequent construction of theoretical models of human NAT1 and NAT2 revealed structural similarities to cysteine proteases (19Sinclair J.C. Sandy J. Delgoda R. Sim E. Noble M.E. Nat. Struct. Biol. 2000; 7: 560-564Crossref PubMed Scopus (188) Google Scholar, 20Rodrigues-Lima F. Deloménie C. Goodfellow G.H. Grant D.M. Dupret J.M. Biochem. J. 2001; 356: 327-334Crossref PubMed Scopus (62) Google Scholar, 21Rodrigues-Lima F. Dupret J.M. Biochem. Biophys. Res. Commun. 2002; 291: 116-123Crossref PubMed Scopus (49) Google Scholar, 22Sandy J. Mushtaq A. Kawamura A. Sinclair J. Sim E. Noble M. J. Mol. Biol. 2002; 318: 1071-1083Crossref PubMed Scopus (102) Google Scholar). These data demonstrated the existence of a conserved cysteine protease-like catalytic triad (Cys, His, and Asp) in NATs (19Sinclair J.C. Sandy J. Delgoda R. Sim E. Noble M.E. Nat. Struct. Biol. 2000; 7: 560-564Crossref PubMed Scopus (188) Google Scholar, 20Rodrigues-Lima F. Deloménie C. Goodfellow G.H. Grant D.M. Dupret J.M. Biochem. J. 2001; 356: 327-334Crossref PubMed Scopus (62) Google Scholar, 21Rodrigues-Lima F. Dupret J.M. Biochem. Biophys. Res. Commun. 2002; 291: 116-123Crossref PubMed Scopus (49) Google Scholar, 22Sandy J. Mushtaq A. Kawamura A. Sinclair J. Sim E. Noble M. J. Mol. Biol. 2002; 318: 1071-1083Crossref PubMed Scopus (102) Google Scholar), confirming the fundamental role in catalysis of a conserved active site cysteine residue (11Pompeo F. Brooke E. Kawamura A. Mushtaq A. Sim E. Pharmacogenomics. 2002; 3: 19-30Crossref PubMed Scopus (61) Google Scholar, 23Dupret J.M. Grant D.M. J. Biol. 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Biochemistry. 1999; 38: 13574-13583Crossref PubMed Scopus (125) Google Scholar, 29Giles N.M. Giles G.I. Jacob C. Biochem. Biophys. Res. Commun. 2003; 300: 1-4Crossref PubMed Scopus (166) Google Scholar). Nitric oxide and reactive nitrogen species (RNS) are major biological oxidants. They have been implicated in major physiological and pathophysiological processes such as vasorelaxation, apoptosis, inflammation, and cancer through the oxidative modification of DNA, proteins, or lipids (30Maeda H. Akaike T. Biochemistry (Mosc.). 1998; 63: 854-865PubMed Google Scholar, 31Ignarro L.J. Biosci. Rep. 1999; 19: 51-71Crossref PubMed Scopus (220) Google Scholar, 32Groves J.T. Curr. Opin. Chem. Biol. 1999; 3: 226-235Crossref PubMed Scopus (177) Google Scholar, 33Liu L. Stamler J.S. Cell Death Differ. 1999; 6: 937-942Crossref PubMed Scopus (169) Google Scholar). Peroxynitrite (ONOO–) is one of the most reactive and, therefore, most deleterious nitric oxide derivatives involved in the oxidative modification of biological molecules (32Groves J.T. Curr. Opin. Chem. Biol. 1999; 3: 226-235Crossref PubMed Scopus (177) Google Scholar, 33Liu L. Stamler J.S. Cell Death Differ. 1999; 6: 937-942Crossref PubMed Scopus (169) Google Scholar). Peroxynitrite affects protein functions by modifying essential reactive thiols or tyrosine residues (32Groves J.T. Curr. Opin. Chem. Biol. 1999; 3: 226-235Crossref PubMed Scopus (177) Google Scholar). For instance, it has been shown to irreversibly inactivate fundamental enzymes such as creatine kinase (34Konorev E.A. Hogg N. Kalyanaraman B. FEBS Lett. 1998; 427: 171-174Crossref PubMed Scopus (137) Google Scholar), tryptophan hydroxylase (35Kuhn D.M. Geddes T.J. J. Biol. Chem. 1999; 274: 29726-29732Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), caspases (36Mohr S. Zech B. Lapetina E.G. Brune B. Biochem. Biophys. Res. Commun. 1997; 238: 387-391Crossref PubMed Scopus (202) Google Scholar), and phosphatases (37Takakura K. Beckman J.S. MacMillan-Crow L.A. Crow J.P. Arch. Biochem. Biophys. 1999; 369: 197-207Crossref PubMed Scopus (191) Google Scholar). XMEs, such as cytochromes P450 (38Daiber A. Herold S. Schoneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar, 39Lin H.L. Kent U.M. Zhang H. Waskell L. Hollenberg P.F. Chem. Res. Toxicol. 2003; 16: 129-136Crossref PubMed Scopus (50) Google Scholar) and glutathione S-transferase (40Wong P.S. Eiserich J.P. Reddy S. Lopez C.L. Cross C.E. van der Vliet A. Arch. Biochem. Biophys. 2001; 394: 216-228Crossref PubMed Scopus (74) Google Scholar), have also been reported to be irreversibly inactivated by peroxynitrite. Peroxynitrite generation has been demonstrated during sepsis as well as in autoimmune and inflammatory conditions (32Groves J.T. Curr. Opin. Chem. 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As stated above, NAT1 has been shown to be highly expressed in normal and cancerous human breast tissue (17Adam P.J. Berry J. Loader J.A. Tyson K.L. Craggs G. Smith P. De Belin J. Steers G. Pezzella F. Sachsenmeir K.F. Stamps A.C. Herath A. Sim E. O'Hare M.J. Harris A.L. Terrett J.A. Mol. Cancer Res. 2003; 1: 826-835PubMed Google Scholar). Therefore, we decided to investigate whether peroxynitrite could inhibit endogenous NAT1 in MCF7 breast cancer cells. Using peroxynitrite and the peroxynitrite generator 3-morpholinosydnonimine N-ethylcarbamide (SIN1), we investigated the effect of peroxynitrite on the activity of NAT1 in human breast cancer cells. We found that exposure of cultured MCF7 cells to physiological concentrations of peroxynitrite or SIN1 for a short period of time (10–30 min) led to the irreversible inactivation of the endogenous NAT1 enzyme. Further mechanistic analyses of the reaction of peroxynitrite with purified recombinant NAT1 showed that this XME is very rapidly (kinact = 5 × 104m–1·s–1) and irreversibly inactivated by physiological concentrations of peroxynitrite. In addition, we showed that the peroxynitrite-dependent inactivation of NAT1 was due to irreversible oxidative modification of the catalytic cysteine residue of the enzyme. Thus, our results suggest that NAT1 activity may be regulated in vivo by nitrosative stress, with potentially important implications in carcinogenesis and tumor progression. Materials—Peroxynitrite and SIN1 hydrochloride were obtained from Calbiochem-Novabiochem, and their chemical structures are shown in Fig. 1. p-Aminosalicylic acid (PAS), p-nitrophenylacetate (PNPA), acetyl-coenzyme A (AcCoA), coenzyme A (CoA), 1,4-dithiothreitol (DTT), and reduced glutathione (GSH) were obtained from Sigma. The vector, pET28, was purchased from Novagen. Nickel-nitrilotriacetic acid Superflow resin was obtained from Qiagen. Anti-fluorescein Fab′ fragments conjugated to peroxidase, fluorescein-conjugated iodoacetamide, and Complete protease inhibitor tablets were obtained from Roche Applied Science. The Bradford protein assay kit was supplied by Bio-Rad. All other reagents were purchased from Sigma or Eurobio. Polyclonal antibody against human NAT1 was a kind gift of Prof. Edith Sim (University of Oxford, Oxford, UK). Cell Culture, Peroxynitrite/SIN1 Treatment, and Total Cell Extracts—MCF7 cells (human breast carcinoma) were cultured as monolayers in 100-mm Petri dishes at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% (v/v) fetal bovine serum and penicillin/streptomycin. At ≈90% confluence, cell monolayers were washed with PBS (Ca2+/Mg2+). Cell monolayers were exposed to different concentrations of peroxynitrite or SIN1 in 10 ml of PBS and kept for 10 min (peroxynitrite) or 30 min (SIN1) at 37 °C. Control treatments were performed with decomposed SIN1 (obtained by allowing decomposition at room temperature in the dark for 48 h) or with PBS only. After treatment, monolayers were washed with PBS, scraped in 1 ml of lysis buffer (20 mm Tris-HCl, 1 mm DTT, pH 7.5, 0.2% Triton X-100, and protease inhibitors) and centrifuged for 1 h at 100,000 × g. Supernatants (total cell extracts) were taken, and protein concentrations were determined using the Bradford method. All cell extracts were adjusted to the same protein concentration by adding 20 mm Tris-HCl and 1 mm DTT, pH 7.5, and then used in the experiments described below. Detection of Endogenous NAT1 Protein in MCF7 Cell Extracts—To assess the amount of NAT1 in MCF7 cell extracts, all NAT1 protein present in these extracts was immunoprecipitated for 2 h at 4 °C using a saturating amount of purified anti-NAT1 antibody (1 μg of IgG fraction) in a total volume of 300 μl. Equal amounts of protein A-agarose were then added, and the mixture was rocked for 2 h at 4 °C. Beads were then washed twice with PBS. Bound NAT1 was eluted by boiling in non-reducing SDS sample buffer and subjected to SDS-PAGE and Western blotting using the anti-NAT1 antibody. Production and Purification of Recombinant Human NAT1—The human NAT1 cDNA was subcloned into pET28. This construct was used to transform BL21 (DE3) bacteria, which were then induced with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside and cultured for 4 h at 37 °C. Bacteria from a 1-liter culture were collected by centrifugation (6,000 × g, 30 min), washed with PBS (phosphate-buffered saline), and centrifuged again (6,000 × g, 30 min). The pellet was resuspended in 40 ml of 50 mm Tris-HCl, pH 8, 150 mm NaCl (lysis buffer) containing lysozyme (1 mg/ml final concentration), and protease inhibitors. Following incubation (1 h at 4 °C), protease inhibitors, DNase I (20 μg/ml final concentration), and 0.2% Triton X-100 (final concentration) were added, and the suspension was incubated for a further 1 h at 4 °C. The lysate was then subjected to sonication on ice (five pulses of 10 s each) and centrifuged (12,000 × g, 30 min). The supernatant was incubated with 1.5 ml of nickel-nitrilotriacetic acid Superflow resin in the presence of 20 mm imidazole for 2 h at 4 °C. The resin was then poured into a column and washed successively with lysis buffer supplemented with 0.2% Triton X-100 and lysis buffer supplemented with 50 mm imidazole. Recombinant NAT1 was eluted with 300 mm imidazole in lysis buffer. Purified NAT1 was reduced by incubation with 10 mm DTT for 10 min at 4 °C and then dialyzed against 25 mm Tris-HCl, pH 7.5, and 1 mm EDTA and stored at –80 °C. SDS-PAGE analysis was carried out at each stage of purification, and protein concentrations were determined with a standard Bradford assay. Enzyme Assay—Detection of NAT1 activity in MCF7 cell extracts was performed as described previously (45Sinclair J.C. Delgoda R. Noble M.E. Jarmin S. Goh N.K. Sim E. Protein Expression Purif. 1998; 12: 371-380Crossref PubMed Scopus (51) Google Scholar) in a total volume of 100 μl. Cell extracts (50 μl) and p-aminobenzoic acid (200 μm) in assay buffer (20 mm Tris-HCl and 1 mm DTT, pH 7.5) were pre-incubated at 37 °C for 5 min. AcCoA (400 μm) was added to start the reaction, and the samples were incubated at 37 °C for different times (up to 30 min). The reaction was quenched with 100 μl of ice-cold aqueous trichloroacetic acid (20% w/v), and the proteins were pelleted by centrifugation for 5 min at 12,000 × g. 4-Dimethylaminobenzaldehyde (DMAB; 800 μl, 5% w/v in 9:1 acetonitrile/water) was added, and the absorbance was measured in 10-mm pathlength cuvettes at 450 nm (Uvikon Spectrophotometer). The amount of the remaining arylamine was determined from a standard curve. All assays were performed in triplicate in conditions such that the initial rates were linear. Enzyme activities were normalized according to the protein concentration of cell extracts and are expressed as percentages of control NAT1 activity (untreated MCF7 monolayers taken as 100% activity). Recombinant NAT1 enzyme activity was determined spectrophotometrically at 410 nm using PNPA as the acetyl donor and PAS as a NAT1-specific arylamine substrate, as described by Mushtaq et al. (46Mushtaq A. Payton M. Sim E. J. Biol. Chem. 2002; 277: 12175-12181Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Briefly, samples (10–20 μl) were assayed in a reaction mixture containing 500 μm PAS (final concentration) in 25 mm Tris-HCl, pH 7.5, and 1 mm EDTA. Reactions were started by adding PNPA to a final concentration of 125 μm. All assays were carried out in a total volume of 1 ml such that the final concentration of NAT1 was always 15 nm. Reactions were incubated for 10 min at 37 °C and then quenched by SDS (1%). p-Nitrophenol, generated by the hydrolysis of PNPA by NAT1 in the presence of PAS, was quantified by measuring absorbance at 410 nm with an ELISA plate reader (Metertech). One enzyme unit was defined as the amount of enzyme giving an A410 of 0.5 per 10 min per milliliter. For the controls, we omitted the enzyme, PNPA, or PAS. All assays were performed in quadruplicate in conditions such that the initial rates were linear. Enzyme activities are expressed as percentages of control NAT1 activity (untreated enzyme taken as 100% activity). The total volume of the enzyme assay (1 ml) resulted in dilution of the various compounds used to a sufficient extent (1 in 50 or 1 in 100) to prevent interference with NAT1 enzyme activity measurements. Effects of Bolus Peroxynitrite and Peroxynitrite Generated by SIN1 on Recombinant NAT1 Activity in the Presence or Absence of Various Chemical Compounds—SIN1 is a chemical agent that mimics the generation of peroxynitrite occurring under physiological conditions by generating both superoxide and nitric oxide (47Singh R.J. Hogg N. Joseph J. Konorev E. Kalyanaraman B. Arch. Biochem. Biophys. 1999; 361: 331-339Crossref PubMed Scopus (126) Google Scholar). These two molecules react together extremely rapidly (at a constant rate close to the diffusion limit), leading to the quantitative formation of peroxynitrite (37Takakura K. Beckman J.S. MacMillan-Crow L.A. Crow J.P. Arch. Biochem. Biophys. 1999; 369: 197-207Crossref PubMed Scopus (191) Google Scholar, 47Singh R.J. Hogg N. Joseph J. Konorev E. Kalyanaraman B. Arch. Biochem. Biophys. 1999; 361: 331-339Crossref PubMed Scopus (126) Google Scholar). Thus, SIN1 is frequently used to generate peroxynitrite in chemical and biological experimental systems (47Singh R.J. Hogg N. Joseph J. Konorev E. Kalyanaraman B. Arch. Biochem. Biophys. 1999; 361: 331-339Crossref PubMed Scopus (126) Google Scholar). We used SIN1 to generate peroxynitrite in all experiments except in the experiment that determined the rate constant for the reaction of peroxynitrite with NAT1. We assessed the effects on NAT1 activity of peroxynitrite and peroxynitrite released by SIN1 by incubating purified NAT1 (1.5 μm final concentration) with various concentrations of bolus peroxynitrite or SIN1 in 25 mm Tris-HCl, pH 7.5, and 1 mm EDTA (total volume of 10 μl) for 10 min at 37 °C. Mixtures were then assayed for NAT1 activity as described above. We investigated the ability of reducing agents to protect NAT1 (1.5 μm final concentration) from the effects of peroxynitrite by carrying out SIN1 treatment in the presence or absence of various concentrations of DTT or GSH and then determining residual NAT1 activity. We assessed reactivation of the SIN1-treated enzyme by reducing agents as follows. NAT1 enzyme (1.5 μm final concentration) was first treated with SIN1 (250 μm final concentration) as described above. It was then incubated for 10 min at 37 °C with various concentrations of DTT or GSH in a total volume of 20 μl, and a NAT1 assay was carried out. Control assays in the conditions described above, with GSH or DTT only, gave 100% NAT1 activity. We assessed the extent to which AcCoA and CoA protected against the SIN1-dependent inactivation of NAT1 as follows. NAT1 (1.5 μm final concentration) was first incubated with various concentrations of AcCoA or CoA (final concentrations of 100 μm to 5 mm) in 25 mm Tris-HCl, pH 7.5, and 1 mm EDTA (total volume of 10 μl) for 5 min at 37 °C. Samples were then incubated with SIN1 (250 μm final concentration) for 10 min at 37 °C in a total volume of 20 μl and assayed. Control assays carried out in the conditions described above, with AcCoA or CoA only, gave 100% NAT1 activity. Kinetic Analysis of the Peroxynitrite-dependent Inactivation of NAT1—NAT1 (1.5 μm final concentration) was incubated with bolus peroxynitrite (final concentrations of 0–55 μm) at 37 °C in 25 mm Tris-HCl, pH 7.5, and 1 mm EDTA for 5 min at 37 °C and assayed for residual activity. We determined the second-order rate constant for the reaction of peroxynitrite and NAT1 (kinact) by carrying out kinetic analysis as described by Radi et al. (48Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar), taking into account the spontaneous degradation of peroxynitrite at pH 7.5 and 37 °C. Under these conditions, changes in peroxynitrite (PN) and NAT1 concentrations as a function of time are expressed as shown in Equations 1 and 2, d[PN]dt=-kinact·[NAT1]·[PN]-kdec·[PN](Eq. 1) d[NAT1]dt=-kinact·[NAT1]·[PN](Eq. 1) where [NAT1] is the concentration of the enzyme, [PN] the concentration in peroxynitrite, kinact the second-order rate constant for the inactivation of NAT1 by peroxynitrite, and kdec the first-order rate constant for the decomposition of peroxynitrite. In our conditions (37 °C, pH 7.5), the value of kdec has been shown to be 0.9 s–1 (48Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar). The value of kinact was calculated by fitting an equation derived by Radi et al. (48Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar) to the data, as shown in Equation 3, ln[NAT1]t[NAT1]i=kobs·[PN]iwherekobs=-kinactkinact·[NAT1]i+kdec(Eq. 1) and where [NAT1]i and [PN]i are the initial concentrations of NAT1 and peroxynitrite, respectively, and [NAT1]t is the final concentration of active NAT1 after a reaction with a given concentration of peroxynitrite. Finally, Equation 3 predicts that a plot of ln of fractional activity of NAT1 versus the initial concentration of peroxynitrite should be linear. The value of kinact is deduced from the slope (kobs). Kinetic data were plotted and fitted using KaleidaGraph version 3.5 (Abelbeck Software). Fluorescein-conjugated Iodoacetamide Labeling of Proteins—Purified NAT1 (1.5 μm final concentration) was incubated with or without (control) various concentrations of SIN1 (from 50 to 500 μm final concentration) in 25 mm Tris-HCl, pH 7.5, and 1 mm EDTA for 10 min at 37 °C. Samples were then incubated with fluorescein-conjugated iodoacetamide (20 μm final concentration) for 10 min at 37 °C and subjected to SDS-PAGE under reducing conditions and Western blotting with anti-fluorescein Fab′ fragments conjugated to peroxidase. Statistical Analysis—Data are expressed as means ± S.D. of three independent experiments performed in quadruplicate. The statistical significance of differences between means was evaluated using Student's t test. The level of significance was set at p = 0.05. Protein Determination, SDS-PAGE, and Western Blotting—Protein concentrations were determined by the Bradford assay (BioRad). Samples for gel electrophoresis were mixed with reducing or non-reducing 4× SDS sample buffer and separated by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250. For Western blotting after separation by SDS-PAGE, proteins were electrotransferred to a nitrocellulose membrane. The membrane was blocked by incubation with Tris-buffered saline/Tween 20 (TBS) supplemented with 5% nonfat milk powder for 1 h. Anti-fluorescein Fab′ fragments conjugated to horseradish peroxidase (1:100000) in TBS were added. The membrane was then incubated for 1 h and washed with TBS. The Supersignal reagent (Pierce) was used for detection. Peroxynitrite and SIN1-generated Peroxynitrite Inactivates NAT1 in Cul