Absence of Nitric-oxide Synthase in Sequentially Purified Rat Liver Mitochondria
2009; Elsevier BV; Volume: 284; Issue: 30 Linguagem: Inglês
10.1074/jbc.m109.003301
ISSN1083-351X
AutoresPriya Venkatakrishnan, Ernesto Nakayasu, Igor C. Almeida, R. Timothy Miller,
Tópico(s)Renin-Angiotensin System Studies
ResumoData, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[14C]arginine to l-[14C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria. Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[14C]arginine to l-[14C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria. Nitric oxide (NO·) 2The abbreviations used are: NO·nitric oxideNOSnitric-oxide synthaseMTisolated and purified mitochondriadMTdenatured MTsuperoxidemtNOSmitochondrial NOSeNOSendothelial NOSiNOSinducible NOSnNOSneuronal NOSNOSrrecombinant NOSBH4(6R)-5,6,7,8-tetrahydro-l-biopterinLTQ-MSlinear ion trap-mass spectrometryCaMcalmodulinHRPhorseradish peroxidaseVDACvoltage-dependent anion channelCATcatalaseBSAbovine serum albuminHPLChigh pressure liquid chromatographyMSmass spectrometryTCl-thiocitrullinel-NNAnitro-l-arginineHP-TLChigh performance-TLCSMPsubmitochondrial particleCOcontrol. 2The abbreviations used are: NO·nitric oxideNOSnitric-oxide synthaseMTisolated and purified mitochondriadMTdenatured MTsuperoxidemtNOSmitochondrial NOSeNOSendothelial NOSiNOSinducible NOSnNOSneuronal NOSNOSrrecombinant NOSBH4(6R)-5,6,7,8-tetrahydro-l-biopterinLTQ-MSlinear ion trap-mass spectrometryCaMcalmodulinHRPhorseradish peroxidaseVDACvoltage-dependent anion channelCATcatalaseBSAbovine serum albuminHPLChigh pressure liquid chromatographyMSmass spectrometryTCl-thiocitrullinel-NNAnitro-l-arginineHP-TLChigh performance-TLCSMPsubmitochondrial particleCOcontrol. is a highly diffusible, hydrophobic, and gaseous free radical (1Ignarro L.J. 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Alternatively, NO· can combine with to produce the highly reactive species peroxynitrite. nitric oxide nitric-oxide synthase isolated and purified mitochondria denatured MT superoxide mitochondrial NOS endothelial NOS inducible NOS neuronal NOS recombinant NOS (6R)-5,6,7,8-tetrahydro-l-biopterin linear ion trap-mass spectrometry calmodulin horseradish peroxidase voltage-dependent anion channel catalase bovine serum albumin high pressure liquid chromatography mass spectrometry l-thiocitrulline nitro-l-arginine high performance-TLC submitochondrial particle control. nitric oxide nitric-oxide synthase isolated and purified mitochondria denatured MT superoxide mitochondrial NOS endothelial NOS inducible NOS neuronal NOS recombinant NOS (6R)-5,6,7,8-tetrahydro-l-biopterin linear ion trap-mass spectrometry calmodulin horseradish peroxidase voltage-dependent anion channel catalase bovine serum albumin high pressure liquid chromatography mass spectrometry l-thiocitrulline nitro-l-arginine high performance-TLC submitochondrial particle control. 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Nitric Oxide. 2006; 14: 162-168Crossref PubMed Scopus (97) Google Scholar) has reviewed the more recent developments in the area of mitochondrial NO· production and discussed some of the shortcomings of certain techniques still being used. In light of this ongoing controversy regarding the presence or absence of a mtNOS, we designed and carefully conducted properly controlled studies to either confirm or refute the existence of any NOS isoform within mitochondria. Ultrapure rat liver mitochondria were isolated using repeated differential centrifugation followed by Percoll gradient purification. Proteomic analyses were then performed using a nano-HPLC-coupled nanospray LTQ-MS. To avoid the interfering factors that are rampant in NO· trapping assays (43Schmidt K. Klatt P. Mayer B. Biochem. J. 1994; 301: 645-647Crossref PubMed Scopus (65) Google Scholar), the NOS-catalyzed conversion of l-[14C]arginine to l-[14C]citrulline was used to probe for NOS activity in mitochondria. Appropriate controls were employed and, for inhibition studies, high concentrations of l-thiocitrulline (TC) (44Narayanan K. Griffith O.W. J. Med. Chem. 1994; 37: 885-887Crossref PubMed Scopus (110) Google Scholar) were used. Additionally, immunochemical analyses were performed with ultrapure mitochondria using nNOS, eNOS, and CaM antibodies. The problems faced with the commonly used techniques in mtNOS studies are discussed. HEPES, l-arginine·HCl, CaCl2·4H2O, bovine brain CaM, (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), NADPH, l-citrulline, EDTA, EGTA, dithiothreitol, iodoacetamide, sucrose, mannitol, acetone, l-thiocitrulline, N(ω)-nitro-l-arginine (l-NNA), monobasic and dibasic sodium phosphate, SDS, alkaline phosphatase-conjugated anti-rabbit secondary antibody, and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse secondary antibodies were purchased from Sigma. Dowex 50W-X8 was obtained from Supelco (Bellefonte, PA). Acetonitrile was purchased from Mallinckrodt-Baker, Inc. (Philipsburg, NJ). Trichloroacetic acid was obtained from EMD Chemicals, Inc. (San Diego). Tris and nitrocellulose membranes were purchased from Bio-Rad. SuperSignal West Pico chemiluminescent substrate was purchased from Pierce. Percoll and 2′,5′-ADP-Sepharose 4B were products of GE Healthcare. Complete protease inhibitor mixture tablets were purchased from Roche Applied Science. Primary antibodies for the mitochondrial outer membrane marker, voltage-dependent anion channel (VDAC), were obtained from Affinity Bioreagents (Golden, CO). Primary antibodies for glucose-regulated protein (GRP 75)/mitochondrial heat shock protein 70 (mt hsp70) and CaM were purchased from Abcam, Inc. (Cambridge, MA). nNOS and eNOS primary antibodies were generous gifts from Bettie Sue Masters, University of Texas Health Science Center, San Antonio. The tubulin antibody was a gift from Sukla Roychowdhary, University of Texas, El Paso. All other chemicals and reagents were from common suppliers and were of the highest grade commercially available. Recombinant nNOS and eNOS, referred to as nNOSr and eNOSr, respectively, were overexpressed in Escherichia coli and purified according to established methodology (45Martasek P. Liu Q. Liu J. Roman L.J. Gross S.S. Sessa W.C. Masters B.S. Biochem. Biophys. Res. Commun. 1996; 219: 359-365Crossref PubMed Scopus (142) Google Scholar, 46Roman L.J. Sheta E.A. Martasek P. Gross S.S. Liu Q. Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8428-8432Crossref PubMed Scopus (244) Google Scholar). Superoxide dismutase (SOD) and catalase (CAT) were purchased from Sigma. The specific activities of SOD and CAT were 2,500–7,000 units/mg protein and ≥10,000 units/mg protein, respectively. All experimental protocols involving animals were approved by University of Texas, El Paso Institutional Animal Care and Use Committee (IACUC). Male Sprague-Dawley (SD) rats (250–300 g; ∼3 months of age) were obtained from Harlan Sprague-Dawley (Houston, TX) and used in all studies. Pure rat liver mitochondria were obtained by repeated differential centrifugation followed by Percoll gradient purification. Expertise for the mitochondrial purification procedure was obtained first hand and adopted from the methodology used in the laboratory of James Geddes, University of Kentucky, Lexington. Initially, 2–4 rats were euthanized, and the entire livers were excised and immersed in ice-cold mitochondrial isolation buffer (MIB) containing mannitol (215 mm), sucrose (75 mm), EGTA (1 mm), HEPES/KOH (20 mm), pH 7.2. In addition, a commercially available serine and cysteine protease-inhibiting complete mixture tablet was included. The liver lobes were blotted, washed 2–3 times with fresh MIB, and minced into small pieces with scissors. The resulting minced pieces of liver were washed with MIB to remove blood. Then 6–8 ml of ice-cold MIB was added to the washed and minced tissue. Portions of tissue samples were placed in a glass Dounce homogenizer. The homogenizer was then immersed in ice, and the tissue was gently homogenized with six complete strokes of a somewhat loose-fitting Teflon pestle at 250 rpm, using a variable speed motorized unit (Glas-Col LLC., Terre Haute, IN). Following homogenization, both differential centrifugation and Percoll gradient fractionation steps were performed using a pre-cooled SM-24 rotor in an RC-5B Sorvall centrifuge at 4 °C. Each sample was centrifuged for 10 min, unless specified otherwise. First, the tissue homogenate (CO) was suspended in ice-cold MIB and centrifuged at 800 × g. After the first 800 × g spin, the white fatty layer covering the supernatant was carefully removed using a lint-free wipe or cotton wool. The 800 × g supernatant (M1) was then collected, and the isolated pellet containing red spots of blood and cellular debris was discarded. M1 was spun at 10,000 × g to obtain a pellet containing mitochondria (M2). M2 was then gently resuspended by homogenization using the same glass Dounce homogenizer as before (four strokes at 250 rpm). The differential centrifugation steps were then repeated with the MIB-resuspended M2 at 800 × g (M3) and 10,000 × g (M4) to obtain a relatively pure mitochondrial preparation. The mitochondrial pellet obtained after the final 10,000 × g spin was resuspended in ice-cold MIB and then centrifuged at the lower speed of 9,000 × g. The pellet (M5) isolated during this step contained highly purified mitochondria. Mitochondria (M5) were further purified using Percoll gradient centrifugation as described previously (47Brown M.R. Sullivan P.G. Geddes J.W. J. Biol. Chem. 2006; 281: 11658-11668Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Percoll was chosen as the gradient medium because of its chemical inertness and negligible osmolarity (48Pertoft H. Laurent T.C. Låås T. Kågedal L. Anal. Biochem. 1978; 88: 271-282Crossref PubMed Scopus (197) Google Scholar, 49Reinhart P.H. Taylor W.M. Bygrave F.L. Biochem. J. 1982; 204: 731-735Crossref PubMed Scopus (70) Google Scholar). Ice-cold MIB without EGTA was used in all steps for preparing the Percoll gradient solutions as well as for the wash steps. Mitochondria were supplemented with an equal volume of 30% Percoll (final concentration, mitochondria (M5) in 15% Percoll). A discontinuous Percoll gradient was used, with the bottom layer containing 40% Percoll, followed by 24% Percoll, and finally by 15% Percoll-containing mitochondria. The density gradient was spun at 30,400 × g, and the band between 24 and 40% Percoll (containing the intact mitochondria) was carefully collected. The broken mitochondria at the bottom of the gradient were discarded. The intact mitochondrial sample was then suspended in MIB without EGTA and centrifuged at 16,700 × g for 15 min. Discarding the supernatant, the resulting loose pellet obtained was resuspended in MIB without EGTA and subsequently centrifuged at 13,000 × g followed by a 10,000 × g spin. The intact mitochondrial pellet obtained after the final 10,000 × g spin was collected and used for experimentation. These intact, pure mitochondria, obtained after repeated differential centrifugation and Percoll gradient fractionation, are referred as "MT" throughout. The protein concentration of the pure mitochondrial sample (MT) was measured using the Bradford protein assay (50Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) using BSA as a standard. Intact MT, were placed in a small glass vial residing within an ice-jacket and sonicated mildly for 20 s, with alternating pulse and pause interval times of 5 s each (amplitude-25%) using an ultrasonic processor (Sonics and Materials, Inc.) equipped with a ⅛-inch diameter probe. For preparation of the positive control, sonication of MT was interrupted after 10 s, and nNOSr (200 fmol) was spiked into the half-sonicated MT sample, and then the spiked samples were sonicated for the remaining 10 s. Submitochondrial particles were subsequently obtained from the experimental as well as the positive control samples by ultracentrifugation at 100,000 × g for 1 h at 4 °C (TLA 100 rotor; Beckman Allegra 64 centrifuge). The supernatant fraction, containing the SMPs, and the solubilized pellet (solubilized using 0.1% octyl β-d-glucopyranoside) were collected for further processing. SMPs, the solubilized pellets of MT, as well as the corresponding positive controls, were subjected to 2′,5′-ADP-Sepharose 4B chromatography to enrich samples with NAD(P)H-binding proteins, including NOS, if present. Briefly, the SMPs and the solubilized pellets obtained from purified MT (initial amounts varied depending on the experiment) were incubated with 150 μl of the 2′,5′-ADP-Sepharose 4B resin slurry, equilibrated with ice-cold sodium phosphate buffer (50 mm, pH 7.4), and spun overnight (15 h) at 4 °C. Mini-columns were fabricated in the laboratory using 200-μl pipette tips and the suspension of SMPs that had been preincubated with 2′,5′-ADP-Sepharose 4B beads. The columns were then washed with ice-cold sodium phosphate buffer (5× 100-μl portions). NAD(P)H-dependent proteins bound to the columns were then eluted using 5× 100-μl portions of 5 mm NADPH in Tris (50 mm) containing NaCl (500 mm), pH 7.4. Protein precipitation was performed by treating the affinity-purified eluates of SMP and the corresponding positive controls with 10% trichloroacetic acid for 30 min on ice. Samples were vortexed every 10 min and finally centrifuged at 16,000 × g for 20 min at 4 °C. The trichloroacetic acid-precipitated samples were resuspended in acetone by repeated up-and-down pipetting and centrifuged at 16,000 × g for 20 min at 4 °C. Sample pellets were then dried using a Centri-Vap (Eppendorf). Dry precipitated proteins were digested as described by Stone and Williams (51Stone K.L. Williams K.R. Walker J.M. The Protein Protocol Handbook. Humana Press Inc., Totowa, NJ1996: 415-425Google Scholar). Briefly, dried pellets from acetone washes were solubilized with a solution of 8 m urea, 0.4 m NH4HCO3, and the sulfhydryls were reduced using dithiothreitol (9 mm) for 15 min at 50 °C. The samples were allowed to cool to room temperature and then treated with iodoacetamide (20 mm) for 15 min at room temperature. Subsequently, the iodoacetamide-treated samples were diluted with water to obtain a final concentration of 1 m urea. Finally, the samples were digested overnight with 1 μg of sequencing grade trypsin (Promega) for every 50 μg of mitochondrial protein. Proteolysis was stopped by adding 1 μl of 100% formic acid (Sigma). The resulting samples were desalted using reverse phase ZipTips manufactured with 200-μl micropipette tips and containing POROS 50 R2 resin (Applied Biosystems) (52Jurado J.D. Rael E.D. Lieb C.S. Nakayasu E. Hayes W.K. Bush S.P. Ross J.A. Toxicon. 2007; 49: 339-350Crossref PubMed Scopus (25) Google Scholar). Next, strong cation exchange chromatography was performed using POROS 50 HS resin (53Rodrigues M.L. Nakayasu E.S. Oliveira D.L. Nimrichter L. Nosanchuk J.D. Almeida I.C. Casadevall A. Eukaryot. Cell. 2008; 7: 58-67Crossref PubMed Scopus (358) Google Scholar), and the samples were fractionated by eluting with increasing concentrations of NaCl (0–500 mm). The eluates were again desalted using reverse phase ZipTips and dried under vacuum centrifugation (Centri-Vap). Each of the desalted strong cation exchange fractions were solubilized in 30 μl of 0.05% trifluoroacetic acid, and 8 μl were injected onto a trap column (C18, 0.25 μl, OPTI-PAK). Separations were performed using a reverse phase capillary column (Acclaim, 3 μm C18, 75 μm × 25 cm, LC Packings, Dionex) connected to a nano-HPLC system (nano-LC 1D plus, Eksigent). For elution, the mobile phases were as follows: A, 2% acetonitrile, 0.1% formic acid; B, 80% acetonitrile, 0.1% formic acid. A linear gradient from 0 to 40% solvent B over 100 min was used. The eluting peptides were directly introduced into a linear ion trap-mass spectrometer equipped with a nanospray source (LTQ XL, Thermo-Fisher Scientific). MS spectra were collected in centroid mode over the range of 400–1700 m/z, and the five most abundant ions were submitted twice to collision-induced dissociation (35% normalized collision energy), before being dynamically excluded for 120 s. All MS/MS spectra were from peptides of 600–4000 Da, and at least 15 fragments were converted into DTA files using Bioworks version 3.3.1 (Thermo-Fisher Scientific). The DTA files were submitted for data base searching using TurboSequest (54Eng J.K. McCormack A.L. Yates J.R. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5315) Google Scholar) (available in Bioworks version 3.3.1) and compared against rat NOS sequences (version 3.25) from the International Protein Index. All sequences were submitted in the forward and reverse orientations for calculating the false-positive rate. The data base search parameters included the following: (i) trypsin cleavage in both peptide termini allowing one missed cleavage site; (ii) carbamidomethylation of cysteine residues as a fixed modification; (iii) oxidation of methionine residues as a variable modification; and (iv) 2.0- and 1.0-Da mass tolerance for peptide and fragment, respectively. The following filters in Bioworks were applied as follows: distinct peptides, consensus scores ≥10.2, DCn ≥ 0.1, protein probability ≤1 × 10−3, and Xcorr ≥ 1.5, 2.0 and 2.5 for singly, doubly, and triply charged peptides, respectively. To ensure the quality of the analyses, the false-positive rate was calculated by dividing the number of hits matching the reverse sequences by the total number of identifications. The conversion of l-[14C]arginine to l-[14C]citrulline was used to estimate NOS activity (55Nishimura J.S. Narayanasami R. Miller R.T. Roman L.J. Panda S. Maste
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