guanosine-diphosphate and lauric-acid

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.

Topics: Adipose Tissue, Brown; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Carrier Proteins; Cricetinae; Guanosine Diphosphate; Ion Channels; Lauric Acids; Membrane Proteins; Mitochondrial Proteins; Proteolipids; Protons; Reproducibility of Results; Spectrophotometry; Uncoupling Protein 1

Effects of cold exposure in vivo and treatment with laurate, carboxyatractylate, atractylate, nucleotides, and BSA in vitro on potato tuber mitochondria have been studied. Cold exposure of tubers for 48-96 h resulted in some uncoupling that could be reversed completely by BSA and partially by ADP, ATP, UDP, carboxyatractylate, and atractylate. UDP was less effective than ADP and ATP, and atractylate was less effective than carboxyatractylate. The recoupling effects of nucleotides were absent when the nucleotides were added after carboxyatractylate. GDP, UDP, and CDP did not recouple mitochondria from either the control or the cold-exposed tubers. This indicates that the cold-induced fatty acid-mediated uncoupling in potato tuber mitochondria is partially due to the operation of the ATP/ADP antiporter. As to the plant uncoupling protein, its contribution to the uncoupling in tuber is negligible or, under the conditions used, somehow desensitized to nucleotides.

Topics: Adaptation, Physiological; Adenosine Diphosphate; Atractyloside; Carrier Proteins; Cold Temperature; Enzyme Inhibitors; Guanosine Diphosphate; Intracellular Membranes; Ion Channels; Lauric Acids; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Oxygen Consumption; Serum Albumin, Bovine; Solanum tuberosum; Uncoupling Protein 1

The roles of mild uncoupling caused by free fatty acids (mediated by plant uncoupling mitochondrial protein (PUMP) and ATP/ADP carrier (AAC)) and non-coupled respiration (alternative oxidase (AO)) on H(2)O(2) formation by plant mitochondria were examined. Both laurate and oleate prevent H(2)O(2) formation dependent on the oxidation of succinate. Conversely, these free fatty acids (FFA) only slightly affect that dependent on malate plus glutamate oxidation. Carboxyatractylate (CAtr), an inhibitor of AAC, completely inhibits oleate- or laurate-stimulated oxygen consumption linked to succinate oxidation, while GDP, an inhibitor of PUMP, caused only a 30% inhibition. In agreement, CAtr completely restores the oleate-inhibited H(2)O(2) formation, while GDP induces only a 30% restoration. Both oleate and laurate cause a mild uncoupling of the electrical potential (generated by succinate), which is then followed by a complete collapse with a sigmoidal kinetic. FFA also inhibit the succinate-dependent reverse electron transfer. Diamide, an inhibitor of AO, favors the malate plus glutamate-dependent H(2)O(2) formation, while pyruvate (a stimulator of AO) inhibits it. These results show that the succinate-dependent H(2)O(2) formation occurs at the level of Complex I by a reverse electron transport. This generation appears to be prevented by mild uncoupling mediated by FFA. The anionic form of FFA appears to be shuttled by AAC rather than PUMP. The malate plus glutamate-dependent H(2)O(2) formation is, conversely, mainly prevented by non-coupled respiration (AO).

Topics: Atractyloside; Carrier Proteins; Glutamic Acid; Guanosine Diphosphate; Hydrogen Peroxide; Ion Channels; Lauric Acids; Malates; Membrane Proteins; Mitochondria; Mitochondrial ADP, ATP Translocases; Mitochondrial Proteins; Oleic Acid; Oxidation-Reduction; Oxidative Phosphorylation; Oxygen Consumption; Pisum sativum; Succinic Acid; Uncoupling Agents; Uncoupling Protein 1