Identification of a Functioning Mitochondrial Uncoupling Protein 1 in 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. 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 by the UCP 1 knock-out mice on a C57BL/6J S. A. Harper Nature. 1997; PubMed Scopus Google Scholar), by mice and in a specific and of brown adipose tissue mitochondria from UCP 1 knock-out and mice, on a C57BL/6J from muscle mitochondrial from UCP knock-out A.J. D. A. J. R. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and mice, on a C57BL/6J from of isolated from Wistar as by F. A. M. Brand M.D. Eur. J. Biochem. Scopus (52) Google Scholar). The thymus was from the of tissue and brown and with and was by through The was The of was by with mouse a mouse on a and using consumption rates by thymocytes isolated from measured in the using isolated from whole mouse whole rat rat rat and rat and mouse isolated by by to the of and and Scholar). isolated from rat muscle by the muscle and the tissue with from the by to the of Biochem. 1991; PubMed Scopus Google Scholar). protein concentrations determined by the of to the of Biochem. PubMed Scopus Google Scholar). and of the RNA was purified from rat BAT, whole rat rat and using RNA was to by using and used to UCP 1 as by and R.K. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). for of the used as a to K. K. K. M. K. S. Google Scholar). as of by of and with a for The on a and and was used to UCP 1 hydroxyapatite chromatography. detected by with D. Scopus Google Scholar). is detect of protein in a and is with the required for mass spectrometry was also used to to UCP purified from in and UCP protein from as by E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar), used as in as by E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar). antibodies to UCP 1 and UCP in by as by E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar). 1 acids and acids from to full-length UCP 1 protein was a from specific for the of from was a from of The used and a the with a in for 1 room using and by to GDP nucleotides from isolated mitochondria using anion by the by Huang and Klingenberg (15Huang S.G. Klingenberg M. Eur. J. Biochem. 1995; 229: 718-725Crossref PubMed Scopus (29) Google Scholar). Isolated mitochondria a of in a 1 with room temperature for 1 of binding of GDP was by a of the by J. Physiol. 1991; PubMed Scopus Google Scholar). with and for in the and presence of GDP binding was from the and The and the binding capacity the binding of obtained by for specific binding sites on the and free concentrations used on the by using the The to a by by consumption rates measured using a oxygen as by M. C. Bouillaud F. Nicholls D.G. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in 1 free BSA, and 1 oxygen consumption rates measured as the rates on addition of The of this oxygen consumption rate to GDP was The of the oxygen consumption rate to was the mitochondrial was to the to the oxygen consumption rate due to The oxygen was to the of B. Biochem. PubMed Scopus Google Scholar) that of oxygen in 1 of of UCP 1 from of UCP 1 was using a hydroxyapatite as by and Klingenberg Klingenberg M. FEBS Lett. 1980; PubMed Scopus Google Scholar) with thymus mitochondria in for The mitochondrial was in in and on for to the mitochondrial The was by of in of for to it a 1 The was from the by for room temperature to of the mitochondrial The the mitochondrial was room temperature for the by of The was for to the (UCP the The protein of the was determined Biochem. PubMed Scopus Google Scholar), and the was using a The was by of in a and to The was from the by for The in of for to and for to The was the with and for with using a The was for and the protein was The protein of this was and of protein by and for protein was from a and was to a and with liquid to any of The was for in of with for with and for with as M. and Scholar). The protein was with of to the from the by using a of with The was to a of in a and for mass spectrometry. mass mapping was using matrix-assisted laser desorption ionization time of flight mass spectrometry. of the was and using a The peptide was from the with 1 of the MALDI-TOF in and a mass in and was used to MALDI-TOF peptide to that peptide to from as and peptide was using The peptide and on a and on a The by tandem mass spectrometry using a mass The as a from to with with to for by a from to for the in the was for it was on for to that from mass was used to of from the the and on to from for to for analysis of protein and protein using UCP 1 transcripts detected in rat thymus and BAT in shown R.K. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar), rat was for UCP 1 The of for of transcripts uncoupling in rat brown adipose tissue, known to of has been R.K. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). Although it was that transcripts for UCP 1 present in rat it was to UCP 1 protein was To that we used 1 polyclonal The of the is in the We showed that UCP 1 protein was present in mitochondria isolated from rat brown adipose tissue and mitochondria isolated from whole rat thymus of and animals in thymocytes from and not UCP 1 was in mitochondria from brown adipose tissue 1). UCP 1 protein was detected in equivalent amounts of mitochondria isolated from rat a for protein to the of was used in on the mitochondrial of for and that was in of the UCP 1 protein in mitochondria isolated from rat thymus compared with the and rat not using other 1 a UCP by E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar) and also to the full-length UCP 1 not The 1 peptide also detected UCP 1 in mitochondria isolated from the thymus of mice but not in mitochondria from thymus of UCP 1 knock-out mice The also of UCP 1 in mitochondria isolated from BAT of mice but not in mitochondria from BAT of UCP 1 knock-out mice as protein was detected using this in mitochondria isolated from of mice of UCP 1 knock-out mice. BAT present in the of the thymus. a is that the of UCP 1 in mitochondria isolated from whole thymus due to BAT of the thymus BAT is from the white thymus it is not However, to was BAT of the a of thymocytes was from thymus from mice. using it was that of in the from and also mice, for the not with the from whole thymus 1 and transcripts and UCP 1 protein detected in this B and UCP 1 is known to contain a purine nucleotide-binding site that is from the side of the mitochondrial inner membrane. in the presence of and purine binding to the purine binding to brown adipose tissue mitochondria used as a of UCP 1 However, from with brown adipose tissue mitochondria from animals room temperature has shown that of the purine nucleotide-binding sites in UCP 1 purine nucleotides already by mitochondria with that purine nucleotides (15Huang S.G. Klingenberg M. Eur. J. Biochem. 1995; 229: 718-725Crossref PubMed Scopus (29) Google Scholar). as in the measured Bmax of binding to brown adipose tissue mitochondria isolated from room temperature to as in The of the binding site for GDP in brown adipose tissue mitochondria not to by as the KD are and using mitochondria from the of we showed that mitochondria UCP 1 from thymus bind in a with equivalent KD and a Bmax to mitochondria from BAT of that room temperature and Furthermore, in rat mitochondria, contain D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (750) Google Scholar, E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar), rat muscle mitochondria contain UCP E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar), and rat mitochondria, contain UCP D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (750) Google Scholar), GDP binding was detected not shown for the that the GDP binding is of the UCP 1 present in thymus mitochondria, as is the for BAT of GDP binding by thymus mitochondria. to Bmax and KD for the GDP binding and BAT mitochondria from rat mitochondria from BAT thymus and muscle Bmax for and BAT mitochondria are KD are and The Bmax and KD for thymus mitochondria are to those for BAT mitochondria and The and the binding capacity the binding of obtained by for specific binding sites on the and the free concentrations used on the by using the The to a by are from in that thymus mitochondria have a UCP it to determined binding of the UCP 1 uncoupling that the nonphosphorylating oxygen consumption rates as of oxygen of mitochondrial of BAT mitochondria from room temperature in the presence of of the and is inhibited by addition of 1 GDP to In the oxygen consumption rate was stimulated by nanomolar amounts of free fatty to to the uncoupled rate of by the addition of In to the UCP 1 in mitochondria from thymus thymocytes from room temperature was we measured the oxygen consumption rates the and we determined rates inhibited by GDP by by In addition, the uncoupled oxygen consumption rate was that oxygen consumption rate by mitochondria was inhibited by GDP and was subsequently stimulated by the presence of a nanomolar of The uncoupled rate is for thymus mitochondria. In oxygen consumption rates by mitochondria from rat contain rat muscle mitochondria contain UCP and rat mitochondria and rat mitochondria not of contain UCP are and was not due to mitochondria as the oxygen consumption rates due to the for mitochondria from liver, and respectively. We also that the oxygen consumption rate was and mitochondria on of not in the of to the mitochondria, as we also that addition of failed to oxygen consumption in mitochondria with not The and GDP binding to that of the UCP 1 protein in mitochondria from rat thymus to used for of UCP 1 from BAT Klingenberg M. FEBS Lett. 1980; PubMed Scopus Google Scholar) for rat thymus. and with a of mass of UCP was The was and for analysis by mass spectrometry. The mass obtained using MALDI-TOF is shown in and for peptide mass the with the peptide identified mitochondrial brown UCP 1 from R. as the and peptide of the UCP 1 protein of identified rat UCP 1 as a major protein in the the was to the and not the other identified protein 1 and mitochondrial from present in amounts those of UCP 1. also identified by of UCP 1 from peptide mass not to on the of the of UCP not to on the of the of UCP 1 in a To the mass of the isolated tandem mass spectrometry was by liquid and and the used to the The are shown in of the identified rat also and identified other mitochondrial voltage-dependent protein 1 and protein. is that the of from to thus present in the protein from the of R. mitochondrial using peptide of identified brown UCP in a The of this was to the of the proton leak in thymus. already the and of for of transcripts uncoupling have been R.K. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). In this we that rat thymus contains UCP 1 The that rat thymus also contains transcripts for UCP and UCP R.K. Biochim. Biophys. Acta. 2004; PubMed Scopus Google Scholar). are in to a previous that failed to detect UCP 1 transcripts in rat thymus using Northern blot analysis (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). The to detect UCP 1 transcripts in that a abundance of UCP 1 in the thymus of room detected by the used in this on uncoupling have the of protein by using Northern blot analysis, UCP detected in tissues D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (750) Google Scholar), UCP protein was detected in a of tissues by analysis C. C. C. E. S. D. Bouillaud F. B. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). in UCP also in animal room with UCP the of UCP 1 transcripts in thymus not have the presence of the UCP 1 protein. To that we the UCP for it and to UCP 1. that the UCP was for UCP 1 protein UCP UCP and other mitochondrial present in mitochondria. of the UCP to mitochondria isolated from rat whole thymus UCP a that was by using a polyclonal 1 for by E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar) and a polyclonal to the full-length UCP 1 protein from Furthermore, we to detect UCP 1 in mitochondria isolated from mouse thymus but not in mitochondria from UCP 1 knock-out mice. the that we UCP 1 in the thymus and that the was not with a mitochondrial protein present in thymus but not in other mitochondria. We have that the presence of UCP 1 in thymus is not of BAT of thymus. BAT in the of the and present it is and Furthermore, in a of UCP 1 transcripts detected by and mitochondria isolated from contain the UCP 1 protein. UCP 1 is present in thymocytes. In this it is that of are known to thymus M.D. 1991; PubMed Scopus Google Scholar) and also UCP 1 in BAT P. Eur. J. PubMed Scopus Google Scholar), we detect in the abundance of UCP 1 in mitochondria isolated from rat thymus. that UCP 1 plays a role in thymus than in The presence of UCP 1 in rat thymus that mitochondria isolated from this bind purine To the of the purine binding we to the mitochondria to any purine that Huang and Klingenberg (15Huang S.G. Klingenberg M. Eur. J. Biochem. 1995; 229: 718-725Crossref PubMed Scopus (29) Google Scholar) have shown that of any purine by the mitochondria with We showed that thymus mitochondria isolated from room temperature bind with and with a Bmax of that determined for BAT mitochondria. binding was detected in mitochondria from tissues as and UCP muscle UCP and not any D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (750) Google Scholar, E. C. R.K. Biochim. Biophys. Acta. 2003; PubMed Scopus Google Scholar, K.S. Brand M.D. Biochem. Soc. Trans. 2001; 29: PubMed Google Scholar). We conclude that the binding in thymus mitochondria was due to UCP 1 protein. The of UCP 1 in BAT mitochondria is to heat by metabolic flux in that tissue. BAT mitochondria have oxygen consumption in vitro, due to UCP 1 proton leak activity that is inhibited by purine nucleotides and by long chain fatty acids evidence for the of UCP 1 protein in thymus mitochondria and the of those mitochondria to bind GDP to thymus mitochondria a UCP proton leak to that been for UCP 1 isolated from Although the oxygen consumption rates in thymus mitochondria a that of BAT mitochondria, was a UCP proton leak inhibited by GDP and by nanomolar palmitate. was GDP-sensitive proton leak in mitochondria contain muscle mitochondria contain UCP mitochondria contain UCP D. Bouillaud F. Biochem. J. 2000; 345: 161-179Crossref PubMed Scopus (750) Google Scholar, K.S. Brand M.D. Biochem. Soc. Trans. 2001; 29: PubMed Google Scholar). We conclude that the and proton leak measured in rat thymus mitochondria is not due to UCP UCP but is due to the activity of UCP 1. The of UCP 1 in rat thymus mitochondria using immunoblotting and the of the of the GDP binding in thymus mitochondria to that UCP 1 protein in thymus mitochondria to We used for of UCP 1 from BAT mitochondria Klingenberg M. FEBS Lett. 1980; PubMed Scopus Google Scholar), and we found that rat thymus mitochondria a on a in with the mass of UCP 1 MALDI-TOF mass spectrometry that the protein was uncoupling protein 1 from rat The peptide showed that UCP 1 protein was by the in the from the analysis the presence of UCP 1 analysis also from other and mitochondrial protein. have of and it is not that with UCP 1 as a on a However, the of the are and from to by a with than UCP UCP was detected in the purified the that UCP UCP through hydroxyapatite M.B. Echtay K.S. Brand M.D. Biochem. J. 2002; PubMed Google Scholar, M. K.D. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). We that the abundance of UCP 1 protein in thymus is than that for UCP UCP The of UCP 1 protein in rat thymus mitochondria has for the and of the thymus. F. A. M. Brand M.D. Eur. J. Biochem. Scopus (52) Google Scholar, S. U. S. A. 2002; PubMed Scopus Google Scholar) have a proton leak a UCP proton in in and we UCP 1 to a major role in metabolic flux in the thymus. is also evidence that UCP 1 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). The of UCP 1 in thymus to suggests a role for UCP 1 in thymus than in The of UCP 1 in rat thymus mitochondria has thus a for thymus and We for UCP and of for the mitochondria isolated from the muscle of and UCP knock-out for the mitochondria isolated from brown adipose tissue of and UCP 1 knock-out and of for the antibodies to the of the with

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