FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2094014080> ?p ?o ?g. }

• W2094014080 endingPage "15543" @default.
• W2094014080 startingPage "15534" @default.
• W2094014080 abstract "We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent Bmax and KD values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus. We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent Bmax and KD values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus. UCP1 (uncoupling protein 1 also known as UCP and thermogenin) has been associated exclusively with brown adipose tissue (BAT) 1The abbreviations used are: BAT, brown adipose tissue; BSA, bovine serum albumin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HTP, hydroxyapatite; LC-MS/MS, liquid chromatography/tandem mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; octyl-POE, octylpentaoctylethylene; RT, reverse transcription; UCP, uncoupling protein; VDAC, voltage-dependent anion channel. 1The abbreviations used are: BAT, brown adipose tissue; BSA, bovine serum albumin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HTP, hydroxyapatite; LC-MS/MS, liquid chromatography/tandem mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time of flight mass spectrometry; octyl-POE, octylpentaoctylethylene; RT, reverse transcription; UCP, uncoupling protein; VDAC, voltage-dependent anion channel. (1Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1338) Google Scholar, 2Nicholls D.G. Biochem. Soc. Trans. 2004; 29: 751-755Crossref Scopus (83) Google Scholar) and is a prerequisite for nonshivering thermogenesis in mammals. UCP 1 is known to transport protons and dissipates the proton electrochemical gradient (Δp) across the mitochondrial inner membrane. UCP 1 thus acts as a major regulator of metabolic flux in mitochondria and as a heat regulator in the whole animal (1Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1338) Google Scholar, 2Nicholls D.G. Biochem. Soc. Trans. 2004; 29: 751-755Crossref Scopus (83) Google Scholar, 3Nicholls D.G. Eur. J. Biochem. 1976; 62: 223-228Crossref PubMed Scopus (208) Google Scholar, 4Ricquier D. Bouillaud F. Trayhurn P. Nicholls D.G. Brown Adipose Tissue. Edward Arnold Publishers, London1986: 86-104Google Scholar). Recent evidence obtained in vitro also suggests that UCP 1 also plays a role in regulating superoxide production by mitochondria (5Echtay K.S. Murphy M.P. Smith R.A.J. Talbot D.A. Brand M.D. J. Biol. Chem. 2002; 277: 47129-47135Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 6Echtay K.S. Roussel D. St-Pierre J. Jekabsons M.B. Cadenas S. Stuart J.A. Harper J.A. Roebuck S.J. Morrison A. Pickering S. Clapham J.C. Brand M.D. Nature. 2002; 415: 96-99Crossref PubMed Scopus (1135) Google Scholar, 7Echtay K.S. Esteves T.E. Pakay J.L. Jekabsons M.B. Lambert A.J. Portero-Otin M. Pamplona R. Vidal-Puig A.J. Wang S. Roebuck S.J. Brand M.D. EMBO J. 2003; 22: 4103-4110Crossref PubMed Scopus (506) Google Scholar, 8Murphy M.P. Echtay K.S. Blaikie F.H. Asin-Cayuela J. Cochemé H.M. Green K. Buckingham J. Taylor E.R. Hurrell F. Hughes G. Miwa S. Cooper C.E. Svistunenko D.A. Smith R.A.J. Brand M.D. J. Biol. Chem. 2003; 278: 48534-48545Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). UCP 1 synthesis, mitochondrial biogenesis, and thermogenesis are controlled by sympathetic nerve activity and thyroid status. Isolated brown adipocytes from cold-acclimated animals have increased oxygen consumption compared with those from room temperature animals, and the mitochondria isolated from active BAT are uncoupled (1Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1338) Google Scholar). Although the mechanism of the uncoupling phenomenon is still a matter of investigation (9Skulachev V.P. FEBS Lett. 1991; 294: 158-162Crossref PubMed Scopus (393) Google Scholar, 10Klingenberg M. Echtay K.S. Bienengraeber M. Winkler E. Huang S.G. Int. J. Obes. Relat. Metab. Disord. 1999; 6: S24-S29Crossref Scopus (61) Google Scholar, 11Garlid K.D. Jabůrek M. Ježek P. Vařecha M. Biochim. Biophys. Acta. 2000; 1459: 383-389Crossref PubMed Scopus (107) Google Scholar), it is known that long chain fatty acids are required for uncoupling activity and that purine nucleotides inhibit uncoupling through UCP 1 in mitochondria and in the reconstituted systems. The purine nucleotide-binding site on UCP 1 in mitochondria faces the cytosolic side of the inner membrane, and in the presence of saturating amounts of purine nucleotides in vitro, oxygen consumption rates due to UCP 1 activity are inhibited. Subsequent addition of nanomolar concentrations of long chain free fatty acids in vitro restores the activity of UCP 1-containing mitochondria, and oxygen consumption rate increases (1Nicholls D.G. Locke R.M. Physiol. Rev. 1984; 64: 1-64Crossref PubMed Scopus (1338) Google Scholar, 4Ricquier D. Bouillaud F. Trayhurn P. Nicholls D.G. Brown Adipose Tissue. Edward Arnold Publishers, London1986: 86-104Google Scholar). UCP 1 transcripts in BAT have been determined by using Northern blot analysis, and the UCP 1 protein has usually been detected by using polyclonal antibodies specific for the full-length protein. In addition, UCP 1 abundance in BAT (12Sundin U. Cannon B. Comp. Biochem. Physiol. B Comp. Biochem. 1980; 65: 463-471Crossref Scopus (88) Google Scholar, 13Huang S-G. Klingenberg M. Biochemistry. 1996; 35: 16806-16814Crossref PubMed Scopus (52) Google Scholar, 14Porter R.K. Biochim. Biophys. Acta. 2001; 1504: 120-127Crossref PubMed Scopus (65) Google Scholar) and the degree of masking (binding sites already occupied) of the purine nucleotide-binding sites (15Huang S.G. Klingenberg M. Eur. J. Biochem. 1995; 229: 718-725Crossref PubMed Scopus (29) Google Scholar) have been measured by binding of labeled GDP to mitochondria. To date, UCP 1 protein has not been found in any tissue other than BAT, although it has been sought in liver, heart, epididymal white adipose tissue, parametrial white adipose tissue, and thigh muscle (4Ricquier D. Bouillaud F. Trayhurn P. Nicholls D.G. Brown Adipose Tissue. Edward Arnold Publishers, London1986: 86-104Google Scholar, 16Ricquier D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (750) Google Scholar); and a previous study, using Northern blot analysis, failed to detect UCP 1 transcripts in thymus (17Nègre-Salvayre A. Hirtz C. Carrera G. Cazenave R. Troly M. Salvayre R. Penicaud L. Casteilla L. FASEB J. 1997; 10: 809-815Crossref Scopus (682) Google Scholar). However, in this study, we have detected UCP 1 transcripts in whole thymus and thymocytes by using RT-PCR. We have detected UCP 1 protein by using immunoblotting in mitochondria isolated from whole thymus and thymocytes. We have purified and identified UCP 1 from thymus mitochondria by using mass spectrometry. We have also shown that mitochondria isolated from thymus bind GDP with kinetics consistent with the presence of UCP 1 and have a GDP-sensitive and fatty acid-dependent proton leak indicative of the presence of UCP 1. Tissue Sources and Isolation of Mitochondria—Wild-type C57BL/6J mice, female CD-1 mice (20–25 g), and female Wistar rats (Rattus norvegicus; 180–200 g) were provided by the BioResources Unit, Trinity College Dublin. UCP 1 knock-out mice on a C57BL/6J background (18Enërback S. Jacobsson A. Simpson E.M. Harper M-E. Kozak L.P. Nature. 1997; 387: 90-94Crossref PubMed Scopus (1070) Google Scholar), originally provided by Dr. Leslie Kozak, were bred in-house. All mice and rats were housed in a specific pathogen-free facility and were fed ad libitum unless otherwise stated. Some samples of brown adipose tissue mitochondria from UCP 1 knock-out and wild-type mice, both on a C57BL/6J background, were gifts from Dr. Jan Nedergaard (Werner-Gren Institute, Stockholm, Sweden). Skeletal muscle mitochondrial samples from UCP 3 knock-out (19Vidal-Puig A.J. Grujic D. Zhang C.Y. Hagen T. Boss O. Ido Y. Szczepanik A. Wade J. Mootha V. Cortright R. Muoio D.M. Lowell B.B. J. Biol. Chem. 2000; 275: 16258-16266Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar) and wild-type mice, both on a C57BL/6J background, were gifts from Dr. Jean-Paul Giacobino (University of Geneva, Switzerland). Thymocytes were isolated from Wistar rats (180–200 g) essentially as described by Buttgereit et al. (20Buttgereit F. Grant A. Muller M. Brand M.D. Eur. J. Biochem. 1994; 15: 513-519Crossref Scopus (52) Google Scholar). The thymus was removed from the rat, trimmed clean of connective tissue and brown fat (if present), and transferred into RPMI 1640 (Invitrogen) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma). A single cell suspension was prepared by passage through 70-μm nylon sieves (Falcon). The thymocyte suspension was washed extensively. The viability of cells was >99% by trypan dye exclusion. Cells were incubated with fluorescein isothiocyanate-conjugated mouse anti-rat Thy-1 (CD90; MRC OX-7; Serotec) or a fluorescein isothiocyanate-conjugated mouse IgG1 isotype control (Pharmingen). Data were collected on a FACScan flow cytometer (BD Biosciences) and analyzed using Cellquest software. Oxygen consumption rates by thymocytes isolated from rats were measured in the aforementioned medium using an Oxygraph Respirometer (Oroboros™, Innsbruck, Austria). Mitochondria isolated from whole mouse thymus, whole rat thymus, rat thymocytes, rat kidney, and rat and mouse liver were isolated by homogenization followed by differential centrifugation according to the procedure of Chappell and Hansford (21Chappell J.B. Hansford R.G. Birnie G.D. Subcellular Components: Preparation and Fractionation. Butterworth, London1972: 77-91Google Scholar). Mitochondria were isolated from rat skeletal muscle by mincing the muscle and treating the tissue with nagarse (protease VII, Sigma). Mitochondria were separated from the homogenate by differential centrifugation according to the procedure of Bhattacharya et al. (22Bhattacharya S.K. Thakar J.H. Johnson P.L. Shanklin D.R. Anal. Biochem. 1991; 192: 344-349Crossref PubMed Scopus (87) Google Scholar). Mitochondrial protein concentrations were determined by the reduction of Folins-Ciocalteau phosphomolybdic-phosphotungstic reagent according to the method of Markwell et al. (23Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5307) Google Scholar). PCR and Analysis of the Products—Total RNA was purified from rat BAT, whole rat thymus, rat Thy-1-positive thymocytes, and liver using Tri Reagent™ (Sigma) containing guanidine thiocyanate. Total RNA (1 μg) was reverse-transcribed to cDNA by using avian myeloblastosis virus-reverse transcriptase and random primers (Promega, Southampton, UK). Primers used to amplify UCP 1 were as described by Carroll and Porter (24Carroll A.M. Porter R.K. Biochim. Biophys. Acta. 2004; 1700: 145-150Crossref PubMed Scopus (19) Google Scholar). Primers for application of the glyceraldehyde-3-phosphate dehydrogenase sequence were used as a housekeeping gene control. Glyceraldehyde-3-phosphate dehydrogenase (GenBank™ accession number M32599) primers were designed according to Tokunga et al. (25Tokunga K. Nakamura Y. Sakata K. Fujimori K. Ohkubo M. Sawada K. Sakiyama S. Cancer Res. 1987; 47: 5616-5619PubMed Google Scholar). PCR amplification conditions were as follows: initial denaturation of 5 min at 95 °C followed by 35 cycles of 45 s at 95 °C, 45 s at 55 °C, and 2 min at 72 °C with a final extension for 10 min at 72 °C. The PCR mixtures were electrophoresed on a 1% (w/v) agarose gel incorporating ethidium bromide and visualized under ultraviolet light. Polyacrylamide Gels and Immunoblot Analysis—One-dimensional SDS-PAGE under reducing conditions was used to examine UCP 1 purity after hydroxyapatite chromatography. Proteins were detected by staining gels with colloidal Coomassie Brilliant Blue G-250 (26Neuhoff V. Arold N. Taube D. Ehrhardt W. Electrophoresis. 1998; 9: 255-262Crossref Scopus (2349) Google Scholar). This stain is very sensitive (can detect 8–10 ng of protein in a single gel band or spot) and is compatible with the post-staining processing required for mass spectrometry (see below). SDS-PAGE (reducing conditions) was also used to separate proteins prior to immunoblot analysis. UCP 2 purified from inclusion bodies expressed in Escherichia coli and UCP 3 protein from yeast, as described by Cunningham et al. (27Cunningham O. McElligott A.M. Carroll A.M. Breen E. Reguenga C. Oliveira M.E.M. Azevedo J.E. Porter R.K. Biochim. Biophys. Acta. 2003; 1604: 170-179Crossref PubMed Scopus (31) Google Scholar), were used as positive controls in immunoblot experiments. Following SDS-PAGE, resolved proteins were transferred onto polyvinylidene difluoride membranes (Immobilon-PSQ; Millipore), as described by Cunningham et al. (27Cunningham O. McElligott A.M. Carroll A.M. Breen E. Reguenga C. Oliveira M.E.M. Azevedo J.E. Porter R.K. Biochim. Biophys. Acta. 2003; 1604: 170-179Crossref PubMed Scopus (31) Google Scholar). Polyclonal antibodies to UCP 1 and UCP 3 peptides were raised in rabbits by Eurogentec (Herstal, Belgium) as detailed by Cunningham et al. (27Cunningham O. McElligott A.M. Carroll A.M. Breen E. Reguenga C. Oliveira M.E.M. Azevedo J.E. Porter R.K. Biochim. Biophys. Acta. 2003; 1604: 170-179Crossref PubMed Scopus (31) Google Scholar). Commercial anti-UCP 1 (amino acids 145–159) and anti-UCP 2 (amino acids 144–157) rabbit antisera were purchased from Calbiochem. A goat antiserum to full-length rabbit UCP 1 protein was a gift from Dr. Daniel Ricquier, CNRS, Meudon, France. A rabbit antiserum specific for the β-subunit of F1-ATP synthase from Neurospora crassa was a gift from Dr. Matt Harmey, Department of Botany, University College Dublin, Ireland. The antisera were all used at 1:1000 dilution. Following blocking and a 1-h primary antibody incubation, the blots were incubated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution) in phosphate-buffered saline, 0.5% (v/v) Tween 20, 5% (w/v) milk powder for 1 h at room temperature. Blots were developed using an ECL detection system (Amersham Biosciences), and immunoreactions were visualized by exposure to Kodak X-Omat LS film. GDP Binding Assay—Endogenous residual bound nucleotides were removed from isolated mitochondria using an anion exchanger (Dowex 21K) by the procedure described by Huang and Klingenberg (15Huang S.G. Klingenberg M. Eur. J. Biochem. 1995; 229: 718-725Crossref PubMed Scopus (29) Google Scholar). Isolated mitochondria at a concentration of 2 mg/ml in a buffer containing 250 mm sucrose, 20 mm HEPES, 1 mm EDTA, pH 8.0, were shaken with Dowex (120 mg/mg protein) at room temperature for 1 h. Measurement of binding of tritiated GDP was performed by a modification of the procedure described by Scarpace et al. (28Scarpace P.J. Bender B.S. Borst S.E. Can. J. Physiol. Pharmacol. 1991; 69: 761-766Crossref PubMed Scopus (14) Google Scholar). Mitochondria (50 μg) were incubated with [3H]GDP (0.1–6.0 μm, 11.0 Ci/mmol) and [14C]sucrose (250 μCi/ml) for 15 min at 37 °C in the absence (total binding) and presence (nonspecific binding) of unlabeled GDP (1.5 mm). Specific binding was calculated from the difference between total and nonspecific binding. The Michaelis dissociation constant (KD) and the maximal binding capacity (Bmax), describing the saturable binding of [3H]GDP, were obtained by fitting mean values for specific binding sites on the y axis and free radioligand concentrations used on the x axis by using the program “Sigma Plot,” version 5 (SPSS Inc. Chicago). The data were fitted to a rectangular hyperbola by nonweighted, nonlinear least squares regression. Oxygen Consumption by Nonphosphorylating Mitochondria—Oxygen consumption rates were measured using a Clark-type oxygen electrode as described by González-Barroso et al. (29González-Barroso M. Fleury C. Bouillaud F. Nicholls D.G. Rial E. J. Biol. Chem. 1998; 273: 15528-15532Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Mitochondria (1 mg/ml) were incubated at 37 °C in medium containing 120 mm KCl, 5 mm HEPES-KOH, pH 7.4, 1 mm EGTA, 16 μm fatty-acid free BSA, 5 μm atractyloside, 5 μm rotenone, and 1 μg/ml oligomycin. Nonphosphorylating (state 4) oxygen consumption rates were measured as the steady-state rates achieved on addition of 7.5 mm succinate (succinate-KOH, pH 7.4). The sensitivity of this state 4 oxygen consumption rate to GDP (1 mm) was then determined. The sensitivity of the resulting oxygen consumption rate to palmitate (in ethanol) (64 μm) (∼40 nm free) was then determined. Finally, the mitochondrial uncoupler cyanide-p-trifluoromethoxyphenylhydrazone (40 nm FCCP) was added to the chamber to determine the maximum oxygen consumption rate attainable due to uncoupling. The Clark-type oxygen electrode was calibrated according to the procedure of Reynafarje et al. (30Reynafarje B. Costa L.E. Lehninger A.L. Anal. Biochem. 1985; 145: 406-418Crossref PubMed Scopus (195) Google Scholar) assuming that 406 nmol of oxygen atoms were dissolved in 1 ml of incubation medium at 37 °C. Purification of UCP 1 from Rat Thymus Mitochondria—Purification of UCP 1 was performed using a hydroxyapatite (HTP) column chromatography procedure as described by Lin and Klingenberg (31Lin C.S. Klingenberg M. FEBS Lett. 1980; 113: 299-303Crossref PubMed Scopus (212) Google Scholar) with slight modification. Intact thymus mitochondria (8–10 mg) suspended in STE buffer (250 mm sucrose, 5 mm Trizma (Tris base), 2 mm EGTA, pH 7.4) were centrifuged at 22,600 × g for 10 min at 4 °C. The mitochondrial pellet was solubilized in 13% (v/v) octylpentaoctylethylene (octyl-POE) ether in STE (total volume ∼500 μl) and incubated on ice for 10 min prior to loading the solubilized mitochondrial proteins onto an HTP column. The column was prepared by soaking 0.34 g of HTP in 10 ml of STE buffer, pH 7.4, at 4 °C, for 6 h prior to pouring it into a 1-ml column (Bio-Rad) (diameter 1 cm, length 6 cm). The soaking solution was removed from the column by centrifugation at 800 × g for 2 min at room temperature immediately prior to loading of the mitochondrial proteins. The HTP column, containing the solubilized mitochondrial proteins, was incubated at room temperature for 10 min (to denature the adenine nucleotide carrier) followed by 25 min of incubation at 4 °C. The column was then centrifuged at 800 × g for 2 min to remove the HTP eluate (UCP 1-enriched fraction), leaving behind the unwanted bound proteins. The protein concentration of the HTP eluate was determined (23Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5307) Google Scholar), and the octyl-POE detergent was removed using a Bio-Bead (Bio-Rad) column. The Bio-Bead column was prepared by placing 2 ml of Bio-Beads (suspended in distilled/deionized H2O) into a 2-ml syringe barrel and allowing them to settle. The H2O was removed from the Bio-Beads by centrifugation at 800 × g for 2 min. The Bio-Beads were equilibrated in ∼2 ml of STE buffer, pH 7.4, for 30 min prior to use and were further centrifuged at 800 × g for 2 min to remove excess STE buffer. The HTP eluate was loaded onto the Bio-Beads with gentle mixing and incubated for 2 h at 4 °C with periodic slight agitation using a vortex-type mixer. The Bio-Bead column was centrifuged at 800 × g for 2 min, and the protein eluate was collected. The protein concentration of this elute was determined, and ∼5–10 μg of protein were analyzed by SDS-PAGE and colloidal Coomassie Brilliant Blue G-250 staining. Sample Preparation for Mass Spectrometry—A 30–33-kDa colloidal Coomassie Brilliant Blue G-250-stained protein band was excised from a one-dimensional SDS-polyacrylamide gel (Fig. 6A) and was transferred to a 1.5-ml Eppendorf microcentrifuge tube (previously autoclaved and rinsed with 50% high pressure liquid chromatography grade methanol to remove any contaminants) containing 100 μl of 20% (w/v) ammonium sulfate. The excised band was de-stained for 2 days in several washes of 50% (v/v) methanol, 5% (v/v) acetic acid, dehydrated with acetonitrile, reduced for 30 min with 10 mm dithiothreitol at 56 °C, and alkylated for 30 min with 100 mm iodoacetamide at 45 °C as described previously (32Kinter M. Sherman N.E. Protein Sequencing and Identification Using Tandem Mass Spectrometry. John Wiley & Sons, Inc., New York2000: 147-165Google Scholar). The carboxyamidomethylated protein band was digested overnight at 37 °C with 40 μl of 20 ng/μl sequencing grade, modified porcine trypsin according to the manufacturer's directions (Promega, Madison, WI). Peptides were extracted from the gel by using a series of elutions with 10% (v/v) formic acid. The resulting eluate pool was reduced to a final volume of 20 μl in a SpeedVac Concentrator (Savant, Hicksville, NY) and processed for mass spectrometry. Peptide Mass Mapping—Peptide mass mapping was performed using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. A 5-μl amount of the trypsin-digested sample was desalted and concentrated using a micro ZipTip μ-C18 (C18 resin; P10, Millipore Corp., Bedford, MA). The peptide mixture was eluted from the ZipTip with 1 μl of the MALDI-TOF matrix, α-cyano-4-hydroxycinnamic acid (10 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid, Sigma), and spotted onto a Voyager stainless steel MALDI plate (Applied Biosystems, Foster City, CA). An Applied Biosystems Voyager DE-STR mass spectrometer (Applied Biosystems, Foster City, CA) running in delayed extraction and reflectron mode was used to acquire MALDI-TOF data. Selected peptide masses were submitted to four on-line search algorithms that use peptide masses to identify proteins from primary sequence data bases as follows: MS-Fit (Protein Prospector software package; San Francisco, CA, prospector.ucsf.edu/), Mascot (Matrix Science, London, UK, www.matrixscience.com/), ProFound (Prowl, Rockefeller University, prowl.rockefeller.edu), and PeptideSearch (EMBL Bioanalytical Research Group, Heidelberg, Germany, www.mann.embl-heidelberg.de/GroupPages/PageLink/peptidesearch/Services/PeptideSearch/FR_PeptideSearchFormG4.html). Liquid Chromatography/Tandem Mass Spectrometry—The remaining peptide digest was analyzed using LC-MS/MS. The peptide samples were first desalted and concentrated on a PepMap C18 precolumn and then separated on a PepMap C18 column (LC Packings-Dionex, The Netherlands) followed by tandem mass spectrometry using a PE Sciex QStar Pulsar i Quadrupole time-of-flight mass spectrometer (Applied Biosystems). The independent data acquisition parameters were as follows: after a 1-s survey scan from 400 to 1500 m/z, peaks with signal intensity over 10 counts with charge state +2 to +4 were selected for MS/MS fragmentation followed by a 2-s MS/MS from 65 to 1800 m/z for the four most intense ions in the survey scan. Once an ion was selected for MS/MS fragmentation, it was put on an exclude list for 180 s to prevent that ion from being gated again. A 4-atomic mass unit peak window was used to avoid gating of masses from the same isotopic cluster during the survey scan. Keratin and porcine trypsin peak masses were put on an exclude list to prevent these ions from being selected for MS/MS analysis. Independent acquisition data were submitted to ProID (proprietary Applied Biosystems software) for bioinformatics analysis of public protein data base (NCBInr) and subsequent protein identification. By using RT-PCR, UCP 1 transcripts were detected in rat thymus and BAT (Fig. 1, lane 4 in each case). As shown previously (24Carroll A.M. Porter R.K. Biochim. Biophys. Acta. 2004; 1700: 145-150Crossref PubMed Scopus (19) Google Scholar), rat liver was negative for UCP 1 transcripts. The specificity of our primers for detection of transcripts encoding uncoupling proteins 1–3 in rat brown adipose tissue, known to express all of these transcripts, has been demonstrated previously (24Carroll A.M. Porter R.K. Biochim. Biophys. Acta. 2004; 1700: 145-150Crossref PubMed Scopus (19) Google Scholar). Although it was clear that transcripts for UCP 1 were present in rat thymus, it was important to know whether UCP 1 protein was expressed. To that end we used an anti-UCP 1 polyclonal antibody (from Calbiochem). The specificity of the antibody (Calbiochem) is demonstrated in the supplemental figure. We showed that UCP 1 protein was present in mitochondria isolated from rat brown adipose tissue (Fig. 2A, lane 1) and mitochondria isolated from whole rat thymus of fed (lane 2) and fasted (lane 3) animals (and in thymocytes from fed and fasted rats; results not shown). UCP 1 was clearly expressed at higher levels in mitochondria from brown adipose tissue (Fig. 2A, lane 1). As expected, no UCP 1 protein was detected in equivalent amounts of mitochondria isolated from rat liver (Fig. 2A, lane 4). As a control for assessing protein loading, an antibody to the F1 β-subunit of ATP synthase was used in immunoblots on the same mitochondrial preparations (Fig. 2B). Collation of data for three separate preparations and experiments demonstrated that there was no statistically significant difference in expression of the UCP 1 protein in mitochondria isolated from rat thymus when compared with the fed and fasted states (Fig. 2C; or rat thymocytes; results not shown). These results were confirmed using other anti-UCP 1 antibodies, namely a UCP 1-specific anti-peptide antibody characterized by Cunningham et al. (27Cunningham O. McElligott A.M. Carroll A.M. Breen E. Reguenga C. Oliveira M.E.M. Azevedo J.E. Porter R.K. Biochim. Biophys. Acta. 2003; 1604: 170-179Crossref PubMed Scopus (31) Google Scholar) and also an antibody to the full-length UCP 1 (results not shown). The anti-UCP 1 peptide antibody also detected UCP 1 in mitochondria isolated from the thymus of wild-type mice (Fig. 3, lane 6) but not in mitochondria from thymus of UCP 1 knock-out mice (Fig. 3, lane 5). The immunoblot also shows detection of UCP 1 in mitochondria isolated from BAT of wild-type mice (Fig. 3, lane 8) but not in mitochondria from BAT of UCP 1 knock-out mice (lane 7) as expected. No protein was detected using this antibody in mitochondria isolated from liver (Fig. 3, lane 2) or kidney (lane 4) of wild-type mice or liver (lane 1) or kidney (lane 3) of UCP 1 knock-out mice. BAT can be present in the vicinity of the thymus. Therefore, a reasonable concern is that the detection of UCP 1 in mitochondria isolated from whole thymus could be due to BAT contamination of the thymus preparation. As BAT is visibly distinguishable from the translucent white thymus when present, it is easily removed (results not shown). However, to formally ensure there was no BAT contamination of the thymus, a single cell suspension of thymocytes was prepared from thymus from rats or mice. By using flow cytometry, it was confirmed that >99% of cells in the resulting thymocyte suspension from rats, and also mice, were positive for the thymocyte-specific marker, Thy-1 (Fig. 4A; data not shown). Consistent with the data from whole thymus (Figs. 1 and 3), both UCP-1 transcripts and UCP 1 protein were detected in this thymocyte preparation (Fig. 4, B and C). UCP 1 is known to contain a purine nucleotide-binding site that is accessible from the cytoplasmic side of the mitochondrial inner membrane. Thus, in the presence of atractyloside/carboxyatractyloside (which inhibits function and prevents purine nucleotide binding to the adenine nucleotide carrier), purine nucleotide binding to brown adipose tissue mitochondria can be used as a measure of UCP 1 abundance. However, experience from working with brown adipose tissue mitochondria taken from animals held at r" @default.
• W2094014080 created "2016-06-24" @default.
• W2094014080 creator A5018437638 @default.
• W2094014080 creator A5041678671 @default.
• W2094014080 creator A5044469294 @default.
• W2094014080 creator A5049647087 @default.
• W2094014080 creator A5076098159 @default.
• W2094014080 creator A5077403345 @default.
• W2094014080 creator A5077626537 @default.
• W2094014080 creator A5082505178 @default.
• W2094014080 date "2005-04-01" @default.
• W2094014080 modified "2023-10-13" @default.
• W2094014080 title "Identification of a Functioning Mitochondrial Uncoupling Protein 1 in Thymus" @default.
• W2094014080 cites W1504202351 @default.
• W2094014080 cites W1849928642 @default.
• W2094014080 cites W1900866157 @default.
• W2094014080 cites W1964313477 @default.
• W2094014080 cites W1965948140 @default.
• W2094014080 cites W1983872288 @default.
• W2094014080 cites W1986120507 @default.
• W2094014080 cites W1986399270 @default.
• W2094014080 cites W1989858587 @default.
• W2094014080 cites W1997574462 @default.
• W2094014080 cites W1999421079 @default.
• W2094014080 cites W2006107871 @default.
• W2094014080 cites W2013200336 @default.
• W2094014080 cites W2026990654 @default.
• W2094014080 cites W2031165402 @default.
• W2094014080 cites W2031423487 @default.
• W2094014080 cites W2031772127 @default.
• W2094014080 cites W2032814494 @default.
• W2094014080 cites W2034482159 @default.
• W2094014080 cites W2036573181 @default.
• W2094014080 cites W2039423749 @default.
• W2094014080 cites W2058012195 @default.
• W2094014080 cites W2062073832 @default.
• W2094014080 cites W2064435294 @default.
• W2094014080 cites W2080314872 @default.
• W2094014080 cites W2082767273 @default.
• W2094014080 cites W2083623533 @default.
• W2094014080 cites W2100151397 @default.
• W2094014080 cites W2104385322 @default.
• W2094014080 cites W2128569391 @default.
• W2094014080 cites W2141950542 @default.
• W2094014080 cites W2156172044 @default.
• W2094014080 cites W2168456227 @default.
• W2094014080 cites W2336343654 @default.
• W2094014080 cites W2910062165 @default.
• W2094014080 doi "https://doi.org/10.1074/jbc.m413315200" @default.
• W2094014080 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15695816" @default.
• W2094014080 hasPublicationYear "2005" @default.
• W2094014080 type Work @default.
• W2094014080 sameAs 2094014080 @default.
• W2094014080 citedByCount "62" @default.
• W2094014080 countsByYear W20940140802012 @default.
• W2094014080 countsByYear W20940140802013 @default.
• W2094014080 countsByYear W20940140802014 @default.
• W2094014080 countsByYear W20940140802015 @default.
• W2094014080 countsByYear W20940140802016 @default.
• W2094014080 countsByYear W20940140802017 @default.
• W2094014080 countsByYear W20940140802018 @default.
• W2094014080 countsByYear W20940140802019 @default.
• W2094014080 countsByYear W20940140802020 @default.
• W2094014080 countsByYear W20940140802021 @default.
• W2094014080 countsByYear W20940140802022 @default.
• W2094014080 crossrefType "journal-article" @default.
• W2094014080 hasAuthorship W2094014080A5018437638 @default.
• W2094014080 hasAuthorship W2094014080A5041678671 @default.
• W2094014080 hasAuthorship W2094014080A5044469294 @default.
• W2094014080 hasAuthorship W2094014080A5049647087 @default.
• W2094014080 hasAuthorship W2094014080A5076098159 @default.
• W2094014080 hasAuthorship W2094014080A5077403345 @default.
• W2094014080 hasAuthorship W2094014080A5077626537 @default.
• W2094014080 hasAuthorship W2094014080A5082505178 @default.
• W2094014080 hasBestOaLocation W20940140801 @default.
• W2094014080 hasConcept C116834253 @default.
• W2094014080 hasConcept C171089720 @default.
• W2094014080 hasConcept C18903297 @default.
• W2094014080 hasConcept C2779406938 @default.
• W2094014080 hasConcept C2780642333 @default.
• W2094014080 hasConcept C28859421 @default.
• W2094014080 hasConcept C55493867 @default.
• W2094014080 hasConcept C70721500 @default.
• W2094014080 hasConcept C86803240 @default.
• W2094014080 hasConcept C95444343 @default.
• W2094014080 hasConceptScore W2094014080C116834253 @default.
• W2094014080 hasConceptScore W2094014080C171089720 @default.
• W2094014080 hasConceptScore W2094014080C18903297 @default.
• W2094014080 hasConceptScore W2094014080C2779406938 @default.
• W2094014080 hasConceptScore W2094014080C2780642333 @default.
• W2094014080 hasConceptScore W2094014080C28859421 @default.
• W2094014080 hasConceptScore W2094014080C55493867 @default.
• W2094014080 hasConceptScore W2094014080C70721500 @default.
• W2094014080 hasConceptScore W2094014080C86803240 @default.
• W2094014080 hasConceptScore W2094014080C95444343 @default.
• W2094014080 hasIssue "16" @default.
• W2094014080 hasLocation W20940140801 @default.
• W2094014080 hasLocation W20940140802 @default.

• next