Effects of N,N′-bis-(4-trifluoromethylphenyl)-urea on isolated plant mitochondria and thylakoid membranes

The N,N'-bis-(4-trifluoromethylphenyl)-urea is demonstrated to be a powerful uncoupler of ATP formation in plant mitochondria and thylakoid membranes. In mitochondria, the full uncoupling effect was obtained at a concentration of 3-mu-M uncoupling agent in the medium, which corresponded to an inner mitochondrial concentration of 7.2 nmol uncoupling agent mg-1 protein. The full uncoupling concentration was displaced from 3 to 0.6-mu-M when bovine serum albumin was omitted from the reaction medium. Up to 10-mu-M, the studied compound acted as a strictly selective uncoupler. At concentrations higher than 10-mu-M, a selective inhibition of electron transfer occurred at the level of the external NADH dehydrogenase of the inner mitochondrial membrane; the I50 value was 25-mu-M. In thylakoid membranes, the full uncoupling effect was obtained for a concentration of 0.6-mu-M uncoupling agent in the medium, which corresponded to a concentration of 25 nmol uncoupling agent mg-1 chlorophyll. The addition of bovine serum albumin (0.1%) to the reaction medium reversed this effect. For concentrations greater than 2-mu-M, inhibition of PSII electron transfer was observed. Full inhibition was obtained at 15-mu-M uncoupling agent and corresponded to 650 nmol uncoupling agent mg-1 chlorophyll. In marked contrast with classical uncouplers such as CCCP (m-chlorophenylhydrazone) or substituted phenols, the N,N'-bis-(4-trifluoromethylphenyl)-urea cannot be classified among the protonophoric class of uncouplers, due to its inability to exchange protons at pHs between 2 and 9. Its uncoupling action must therefore be attributed either to conformational changes of a transmembrane protein present both in mitochondria and in thylakoid membranes, or to a modification of the arrangement of the phospholipid membrane bilayers.