title: Detection of UCP1 protein and measurements of dependent GDP-sensitive proton leak in non-phosphorylating thymus mitochondria.
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.
authors.affiliation: School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Pearse Street, Dublin 2, Ireland.
authors.forename: Kieran J
authors.initials: KJ
authors.lastname: Clarke
authors.forename: Audrey M
authors.initials: AM
authors.lastname: Carroll
authors.forename: Gemma
authors.initials: G
authors.lastname: O'Brien
authors.forename: Richard K
authors.initials: RK
authors.lastname: Porter
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006153:Guanosine Diphosphate / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000378:metabolism; D015151:Immunoblotting / Q000379:methods*; D007473:Ion Channels / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D010101:Oxygen Consumption / Q000187:drug effects; D011522:Protons; D051381:Rats; D017208:Rats, Wistar; D013950:Thymus Gland / Q000166:cytology*; D000071256:Uncoupling Protein 1
chemical_list: D007473:Ion Channels; D024101:Mitochondrial Proteins; D011522:Protons; C000604373:Ucp1 protein, rat; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D006861:Hydrogen Peroxide