Fernández M, Nicholls D G, Rial E
Eur J Biochem. 1987 May 4;164(3):675-80. doi: 10.1111/j.1432-1033.1987.tb11179.x.
Mitoplasts prepared from brown adipose tissue mitochondria were treated with chymotrypsin and the fragments derived from the 32-kDa uncoupling protein identified by immunoblotting. Extensive proteolysis of the uncoupling protein occurred, the polypeptide pattern being affected by binding of the inhibitory nucleotide GDP. Chymotrypsin modifies the nucleotide binding site, lowering its affinity from 1.7 microM to 21 microM but without decreasing its binding capacity. Nucleotide bound to the modified site can still inhibit the permeation of H+ and Cl- through the protein. The ion conducting pathway itself is also sensitive to chymotrypsin, Cl- and H+ transport being partially inhibited in parallel. The ability of fatty acids to increase the H+ permeability of the protein is also inhibited in parallel with the basal H+ permeability. The results confirm that the transport of H+ and Cl-, and the fatty acid regulation of H+ permeation all share a common structural element within the 32-kDa protein.
