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 dem onstrated to function similarly to the uncoupling protein of brown adipose tissue. In this work, green tomato fruit mitochondria were purified on a se lf-generating Percoll gradient in the presence of 0.5% bovine serum albumin to deplete mitochondria of endogenous free fatty acids. The uncoupling pro tein activity was induced by the addition of linoleic acid during the resti ng state, and in the progressively uncoupled state, as well as during phosp horylating respiration in the presence of benzohydroxamic acid, an inhibito r of the alternative oxidase and with succinate (+ rotenone) as oxidizable substrate. Linoleic acid strongly stimulated the resting respiration in fat ty acid-depleted mitochondria but had no effect on phosphorylating respirat ion, suggesting no activity of the uncoupling protein in this respiratory s tate. Progressive uncoupling of state 4 respiration decreased the stimulati on by linoleic acid. The similar respiratory rates in phosphorylating and f ully uncoupled respiration in the presence and absence of linoleic acid sug gested that a rate-limiting step on the dehydrogenase side of the respirato ry chain was responsible for the insensitivity of phosphorylating respirati on to Linoleic acid. Indeed, the ADP/O ratio determined by ADP/O pulse meth od was decreased by linoleic acid, indicating that uncoupling protein was a ctive 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 d ue to a pure protonophoric activity without any direct effect on the electr on transport chain.