Scatchard analysis of specific guanosine-diphosphate-([3H]GDP-) binding to rat brown-adipose-tissue mitochondria revealed two distinct binding sites with apparent dissociation constants (Kd) of approximately 0.05 and 2.0 μM. Binding to both sites was insensitive to atractyloside. Reducing the pH of the binding medium from 7.1 to 6.6 caused marked reductions in the Kd of both sites, but at pH 7.6, the dissociation constants were increased about 3-fold. Acute treatment of rats with noradrenaline, 1 h before sacrifice, increased the maximum number of binding sites (Bmax, pmol/rng mitochondrial protein) of both sites and also increased the dissociation constants. The Bmax of the lower-affinity site was elevated in rats exposed to 5°C or fed a palatable cafeteria diet for 10 d, compared to control animals, with the greater changes occurring in the cold-adapted group. The high-affinity site was unaltered by cold adaptation or cafeteria feeding. These results indicate the presence of two distinct nucleotide-binding sites in brown-fat mitochondria, both of which may be involved in thermogenesis.

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