Brown‐Adipose‐Tissue Mitochondria: Photoaffinity Labelling of the Regulatory Site of Energy Dissipation

Abstract
Brown-adipose-tissue mitochondria possess an energy-dissipating ion uniport which is inhibited by purine nucleotides. The regulatory nucleotides bind to a high-affinity site on the outer face of the inner membrane which is independent of the adenine nucleotide translocator. A direct correlation between affinity for the regulatory site and ability to inhibit the ion uniport is demonstrated for a number of nucleotide analogues. 8-Azido-adenosine 5′-triphosphate, a photoaffinity label, also competes with GDP for the binding site and induces respiratory control. 8-Azido-adenosine [γ-32P]triphosphate was prepared and covalently bound to hamster brown-adipose-tissue mitochondria by near-ultraviolet irradiation. Two major radioactive bands were identified of apparent molecular weight 30 000 and 32 000, representing 6% and 10% of the inner membrane protein respectively. Selective labelling enabled the 30 000-Mr protein to be identified as the carboxyatractylate binding component of the adenine-nucleotide translocator and the 32 000-Mr protein to be identified as the regulatory site of the energy-dissipating ion uniport. The levels of the 32 000-Mr protein in the inner membrane of guinea-pig brown-adipose-tissue mitochondria correlate with the degree of thermogenic adaptation of the animal.