Effect of hydrophobic sulphydryl reagents on the uncoupling protein and inner-membrane anion channel of brown-adipose-tissue mitochondria.

The effects of three sulphydryl reagents of differing hydrophobicity (N-ethylmaleimide, N-benzylmaleimide and N,N'-o-phenylenedimaleimide) on ion permeation through the inner membrane of brown-adipose-tissue mitochondria are investigated. GDP-sensitive permeation of chloride and protons (hydroxyl ions) through the uncoupling protein is increased exponentially with time by all three reagents. With increasing hydrophobicity of the reagents, modification is enhanced and an initial inhibited state becomes apparent. Results are interpreted in terms of a two-stage modification via a non-transporting intermediate, which does not bind GDP, to a final highly conducting product. The reagents also react with a hydrophilic sulphydryl group on an independent protein to induce a GDP-insensitive pathway which allows chloride, phosphate and sulphate to cross the membrane. The use of different sulphydryl reagents allows the two pathways to be clearly distinguished.