EFFECT OF PH AND MGCL2, ON THE BINDING OF PURINE NUCLEOTIDES TO THE UNCOUPLING PROTEIN IN MEMBRANE-PARTICLES FROM BROWN FAT MITOCHONDRIA

Binding of purine nucleotides to the uncoupling protein (UCP) was inve stigated in membrane particles prepared from brown fat mitochondria of cold-acclimated rats. Mitochondrial membranes were separated from sol uble protein with Lubrol WX and treated with 3 M urea at basic pH. The resulting membrane vesicles were permeable to GDP and contained up to 3 nmol UCP/mg protein with unchanged nucleotide binding, as compared to the mitochondria (GDP/UCP ratio = 1.0; pK(d) GDP = 6.0 at pH 7.0). UCP bound nucleotides to one type of specific binding sites, located e xclusively on the cytosolic side of the mitochondrial membrane. The bi nding affinity of guanine nucleotides was 3-18-times higher than that of the corresponding adenine nucleotides, when measured in membrane pa rticles from cold-acclimated rats, hamsters, and guinea pigs. The pH-d ependent binding affinities of GDP and ADP attained a maximum at FH 5. 0-6.0 (pK(d) GDP = 6.8, pK(c) ADP = 5.8) and were decreased by a facto r of 10(2) at pH 4.0 and pH 8.0, respectively, whereas the binding aff inity of ATP was maximal at pH 1.0 (pK(d) = 7.0) and was decreased by a factor of 10(3) at pH 7.5. Participation of the protein binding cent er in nucleotide interaction with UCP in the membrane was highly pH-de pendent. Mg2+ modified the number of binding sites engaged at a given nucleotide concentration by complex binding of nucleotides; the K-d fo r Mg GTP(2-) and Mg GDP(-) was 20-50-times lower than that of the free nucleotides.