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

TLDR

Brown‑adipose‑tissue mitochondria contain an energy‑dissipating ion uniport that is inhibited by purine nucleotides binding to a high‑affinity site on the outer face of the inner membrane, independent of the adenine nucleotide translocator. The authors used 8‑azido‑adenosine 5′‑triphosphate labeled with γ‑32P and near‑ultraviolet irradiation to covalently attach the probe to hamster brown‑adipose‑tissue mitochondria. Photoaffinity labeling revealed two major proteins (≈30 kDa and 32 kDa); the 30 kDa protein is the carboxyatractylate‑binding component of the adenine‑nucleotide translocator, while the 32 kDa protein is the regulatory site of the ion uniport, with its abundance correlating with thermogenic adaptation and its affinity for the site predicting inhibition potency.

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 [gamma-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 30000 and 32000, representing 6% and 10% of the inner membrane protein respectively. Selective labelling enabled the 30000-Mr protein to be identified as the carboxyatractylate binding component of the adenine-nucleotide translocator and the 32000-Mr protein to be identified as the regulatory site of the energy-dissipating ion uniport. The levels of the 32000-Mr protein in the inner membrane of guinea-pig brown-adipose-tissue mitochondria correlate with the degree of thermogenic adaptation of the animal.