Effect of acclimation temperature on the concentration of uncoupling protein and GDP binding in rat brown fat mitochondria.

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 degrees C and 1.1% in mitochondria from rats acclimated to 29 degrees C. No UCP was found in liver mitochondria. The molar ratio of bound GDP and UCP was 0.5 in mitochondria from warm-acclimated rats and 1.0 in cold-acclimated rats. The GDP/UCP ratio was increased from 0.5 to 1.0 after a 90-min exposure to 4 degrees of warm-acclimated rats; it was decreased from 1.0 to 0.5, when cold-acclimated rats were transferred to 29 degrees C for 2 4h. Treatment of mitochondrial membranes from warm-acclimated rats with 3 M urea at pH 10.0 increased the GDP/UCP 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.