Assessment of a high-throughput screening methodology for the measurement of purified UCP1 uncoupling activity.
Three mitochondrial uncoupling proteins (UCP1, 2, 3) have been described. The proton transport activity of UCP1 triggers mitochondrial uncoupling and thermogenesis but the roles of UCP2 and UCP3 remain debated. Accordingly, compounds able to finely control the proton permeability of the mitochondrial inner membrane where and when needed may have enormous practical consequences. Using purified hamster brown adipose tissue UCP1 reconstituted in liposomes, we describe herein a robust assay allowing the measurement of this artificial membrane conductance to protons in a format compatible with high-throughput screening. The assay was initially developed with a known chemical protonophore in an aproteic system. Then, using the proteolipid reconstituted UCP1 preparation, we assessed the assay with known modulators of UCP1, particularly retinoic acid and guanosine 5'-triphosphate. The system was developed for a 96-well plate format. We then exemplified its use by generating primary data on a set of compounds screened in this system. These primary data will open new routes for the search of candidate compounds that will help biochemical studies on UCPs.
Mozo
Julien
J
BIOTRAM, CNRS UPR-9078, Faculté de Médecine René Descartes Paris 5, site Necker, 156 rue de Vaugirard, 75730 Paris, France.
Ferry
Gilles
G
Masscheleyn
Sandrine
S
Miroux
Bruno
B
Boutin
Jean A
JA
Bouillaud
Frédéric
F
D002001:Adipose Tissue, Brown / Q000737:chemistry; D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D006224:Cricetinae; D006153:Guanosine Diphosphate / Q000494:pharmacology; D007473:Ion Channels; D007850:Lauric Acids / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D024101:Mitochondrial Proteins; D011510:Proteolipids / Q000737:chemistry* / Q000187:drug effects; D011522:Protons; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D007473:Ion Channels; D007850:Lauric Acids; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D011510:Proteolipids; D011522:Protons; D000071256:Uncoupling Protein 1; C020485:proteoliposomes; C030358:lauric acid; D006153:Guanosine Diphosphate; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone