The effect of the acclimation temperature on the concentration of the uncoupling protein (UCP) and specific GDP binding in rat brown adipose tissue mitochondria was investigated. UCP was measured by competition ELISA using purified UCP as a standard and antiserum developed against the C-terminus of the protein. UCP was purified by means of specific polyclonal antibodies immobilized on protein-A-agarose. It represented 2.4% of the total protein in brown fat mitochondria from rats acclimated to 4°C and 1.1% in mitochondria from rats acclimated to 29°C. No UCP was found in liver mitochondria. The molar ratio of bound GDP and UCP was 0.5 in mitochondria from warmacclimated rats and 1 .O in cold-acclimated rats. The GDPKJCP ratio was increased from 0.5 to 1 . O after a 90-min exposure to 4°C of warm-acclimated rats; it was decreased from 1.0 to 0.5, when cold-acclimated rats were transferred to 29°C for 24 h. Treatment of mitochondrial membranes from warm-acclimated rats with 3 M urea at pH 10.0 increased the GDPAJCP ratio from 0.5 to 1.0. Specific GDP binding was a direct measure of the UCP concentration during maximally activated or inactivated thermogenesis in brown adipose tissue. We suggest that variable GDP binding reflects the functional activity of UCP based on different protein conformations.The uncoupling protein (UCP) [I] in brown adipose tissue mitochondria, also called thermogenin [2], allows for a transmembrane flow of protons and thus for a chemiosmotic short circuit that uncouples respiration from ATP synthesis and dissipates chemical energy into metabolic heat. Nucleotides like ATP, ADP, GTP and GDP, which are specifically bound by UCP, inhibit the proton flow and restore respiratory control in the isolated mitochondria (reviewed in [3, 41). Nucleotide interaction with UCP is also likely to regulate the protein function in vivo. UCP, isolated with Triton X-100, has been shown to bind one nucleotide molecule/protein dimer [5]. It is, however, unclear, if this relationship applies also to UCP in the mitochondrial membrane. The molar ratio of bound GDP and UCP in rat brown adipose tissue mitochondria reported in the literature varies extensively [6-111. The relationship is made even more unclear by the altered nucleotide binding observed in mitochondria with unchanged UCP concentrations in response to acute cold or other forms of stimulation of the sympathetic nervous system (reviewed in [12]). According to the latter finding, the stoichiometry of nucleotide binding by UCP corresponds to the thermogenic activity of brown adipose tissue and thus to the activity of UCP. From this point of view, knowledge of the exact relationship between UCP and specifically bound nucleotide seemed important with respect to the molecular mechanism of UCP function. It was the purpose of this study to clarify the stoichiometry of GDP binding by UCP in mitochondria from brown adipose tissue of rats exposed to varying environmental temperatures. MATERIALS AND METHODS AnimalsTen-week-old male Sprague Dawley rats were maintain...