Linoleic acid-induced activity of plant uncoupling mitochondrial protein in purified tomato fruit mitochondria during resting, phosphorylating, and progressively uncoupled respiration.
An uncoupling protein was recently discovered in plant mitochondria and demonstrated to function similarly to the uncoupling protein of brown adipose tissue. In this work, green tomato fruit mitochondria were purified on a self-generating Percoll gradient in the presence of 0.5% bovine serum albumin to deplete mitochondria of endogenous free fatty acids. The uncoupling protein activity was induced by the addition of linoleic acid during the resting state, and in the progressively uncoupled state, as well as during phosphorylating respiration in the presence of benzohydroxamic acid, an inhibitor of the alternative oxidase and with succinate (+ rotenone) as oxidizable substrate. Linoleic acid strongly stimulated the resting respiration in fatty acid-depleted mitochondria but had no effect on phosphorylating respiration, suggesting no activity of the uncoupling protein in this respiratory state. Progressive uncoupling of state 4 respiration decreased the stimulation by linoleic acid. The similar respiratory rates in phosphorylating and fully uncoupled respiration in the presence and absence of linoleic acid suggested that a rate-limiting step on the dehydrogenase side of the respiratory chain was responsible for the insensitivity of phosphorylating respiration to linoleic acid. Indeed, the ADP/O ratio determined by ADP/O pulse method was decreased by linoleic acid, indicating that uncoupling protein was active during phosphorylating respiration and was able to divert energy from oxidative phosphorylation. Moreover, the respiration rates appeared to be determined by membrane potential independently of the presence of linoleic acid, indicating that linoleic acid-induced stimulation of respiration is due to a pure protonophoric activity without any direct effect on the electron transport chain.
Jarmuszkiewicz
W
W
Department of Bioenergetics, Adam Mickiewicz University, Fredry 10, 61-701 Poznan, Poland.
Almeida
A M
AM
Sluse-Goffart
C M
CM
Sluse
F E
FE
Vercesi
A E
AE
D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects*; D006877:Hydroxamic Acids / Q000494:pharmacology; D007473:Ion Channels; D019787:Linoleic Acid / Q000494:pharmacology*; D018551:Solanum lycopersicum; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000187:drug effects*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D010101:Oxygen Consumption / Q000187:drug effects*; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism; D014475:Uncoupling Agents / Q000378:metabolism*; D000071256:Uncoupling Protein 1
D002352:Carrier Proteins; D006877:Hydroxamic Acids; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D010940:Plant Proteins; D014475:Uncoupling Agents; D000071256:Uncoupling Protein 1; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D019787:Linoleic Acid; C006696:benzohydroxamic acid