title: Photoactivated azido fatty acid irreversibly inhibits anion and proton transport through the mitochondrial uncoupling protein.
abstract: The protonophoretic function of uncoupling protein (UCP) is activated by fatty acids. According to the "docking site" hypothesis (Jezek, P., and Garlid, K. D., J. Biol. Chem. 265, 19303-19311, 1990), the fatty acid binding site is identical with the anion channel of UCP. Skulachev (Skulachev, V. P. (1991) FEBS Lett. 294, 158-162) extended this hypothesis by suggesting that fatty acid anions are transported by UCP and that H+ are delivered by back-diffusion of the protonated fatty acid through the lipid bilayer. In this model, UCP does not transport H+ at all but rather enables fatty acids to act as cycling protonophores. New evidence supports this mechanism (Garlid, K. D., Orosz, D. E., Modriansky, M., Vassanelli, S., and Jezek, P. (1996) J. Biol. Chem. 271, 2615-2620). To help elucidate these hypotheses, we synthesized a photoreactive analog of dodecanoic acid, 12-(4-azido-2-nitrophenylamino)dodecanoic acid (AzDA), and studied its effect on transport in mitochondria and proteoliposomes. AzDA behaved in every respect like a typical fatty acid. In micromolar doses, AzDA activated H+ translocation and inhibited Cl- and hexanesulfonate uniport through UCP. After UV light exposure, however, activation of H+ transport was inhibited, whereas inhibition of anion transport was preserved. These effects were irreversible. Photolabeling of mitochondria with [3H]AzDA resulted in a prominent 32 kDa band of UCP, and few other proteins were labeled. The results indicate that AzDA can be ligated to the protein at or near the docking site, causing irreversible inhibition of both H+ and anion transport. The finding that fatty acid-induced H+ transport disappears along with anion transport supports the fatty acid-protonophore mechanism of H+ transport by UCP.
authors.affiliation: Department of Membrane Transport Biophysics, Czech Institute of Physiology, Academy of Sciences, Prague, Chez Republic.
authors.forename: P
authors.initials: P
authors.lastname: Jezek
authors.forename: J
authors.initials: J
authors.lastname: Hanus
authors.forename: C
authors.initials: C
authors.lastname: Semrad
authors.forename: K D
authors.initials: KD
authors.lastname: Garlid
mesh_terms: D002001:Adipose Tissue, Brown / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D000838:Anions; D001665:Binding Sites; D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D066298:In Vitro Techniques; D007473:Ion Channels; D017136:Ion Transport / Q000187:drug effects; D007850:Lauric Acids / Q000378:metabolism / Q000494:pharmacology* / Q000528:radiation effects; D008565:Membrane Proteins / Q000187:drug effects* / Q000378:metabolism; D008647:Mesocricetus; D024101:Mitochondrial Proteins; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000494:pharmacology; D010777:Photochemistry; D011510:Proteolipids; D011522:Protons; D014466:Ultraviolet Rays; D000071256:Uncoupling Protein 1
chemical_list: C098506:12-(4-azido-2-nitrophenylamino)dodecanoic acid; D000838:Anions; D002352:Carrier Proteins; D002712:Chlorides; D007473:Ion Channels; D007850:Lauric Acids; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010169:Palmitic Acids; D011510:Proteolipids; D011522:Protons; D000071256:Uncoupling Protein 1; C020485:proteoliposomes; D019308:Palmitic Acid