Abstract
Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.
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References
Klaus S, Casteilla L, Bouillaud F, Ricquier D (1991) The uncoupling protein UCP: a membraneous mitochondrial ion carrier exclusively expressed in brown adipose tissue. Int J Biochem 23:791–801
Carroll AM, Haines LR, Pearson TW, Brennan C, Breen EP, Porter RK (2004) Immunodetection of UCP1 in rat thymocytes. Biochem Soc Trans 32:1066–1067
Carroll AM, Haines LR, Pearson TW, Fallon PG, Walsh CM, Brennan CM, Breen EP, Porter RK (2005) Identification of a functioning mitochondrial uncoupling protein 1 in thymus. J Biol Chem 280:15534–15543
Porter RK (2006) A new look at UCP 1. Biochim Biophys Acta 1757:446–448
Adams AE, Hanrahan O, Nolan DN, Voorheis HP, Fallon P, Porter RK (2008) Images of mitochondrial UCP 1 in mouse thymocytes using confocal microscopy. Biochim Biophys Acta 1777:115–117
Adams AE, Kelly OM, Porter RK (2010) Absence of mitochondrial uncoupling protein 1 affects apoptosis in thymocytes, thymocyte/T-cell profile and peripheral T-cell number. Biochim Biophys Acta 1797:807–816
Clarke KJ, Adams AE, Manzke LH, Pearson TW, Borchers CH, Porter RK (2012) A role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial uncoupling protein 1 (UCP1). Biochim Biophys Acta 1817:1759–1767
Clarke KJ, Porter RK (2013) Uncoupling protein 1 dependent reactive oxygen species production by thymus mitochondria. Int J Biochem Cell Biol 45:81–89
Chappell JB, Hansford RG (1972) In: Birnie GD (ed) Subcellular components: preparation and fractionation. Buttersworths, London, pp 71–91
Dlasková A, Clarke KJ, Porter RK (2010) The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue. Biochim Biophys Acta 1797:1470–1476
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Markwell MA, Haas SM (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
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Clarke, K.J., Carroll, A.M., O’Brien, G., Porter, R.K. (2015). Detection of UCP1 Protein and Measurements of Dependent GDP-Sensitive Proton Leak in Non-phosphorylating Thymus Mitochondria. In: Palmeira, C., Rolo, A. (eds) Mitochondrial Regulation. Methods in Molecular Biology, vol 1241. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1875-1_11
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DOI: https://doi.org/10.1007/978-1-4939-1875-1_11
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