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‐32 P]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.