SLOW-PHASE KINETICS OF NUCLEOTIDE-BINDING TO THE UNCOUPLING PROTEIN FROM BROWN ADIPOSE-TISSUE MITOCHONDRIA

The kinetics of nucleotide binding to the uncoupling protein (UCP) fro m brown adipose tissue mitochondria were studied with a filter binding method, Fast and dow phases of binding were observed, corresponding t o the two-stage binding model based on equilibrium binding studies (Hu ang, S. G., and Klingenberg, M. (1996) Biochemistry 35, 7846-7854) (Re action 1). [GRAPHICS] REACTION 1 Although this method determines total binding, only the slow phase can be resolved. The fast unresolved pha se represents the formation of the initial loose UCP-nucleotide comple x (UN; K-d approximate to 2 mu M), whereas the slow phase reflects the tight binding (UN) associated with a conformational change induced b y the bound nucleotide, Best fits of the binding data yielded, for the slow phase, k(+1) values of 3.0 x 10(-3) s(-1) for GTP, 4.8 x 10(-3) s(-1) for ATP, 0.13 s(-1) for GDP, and >0.7 s(-1) for ADP and dissocia tion rate constants (k(-1)) of 0.10 x 10(-3) s(-1) for GTP, 0.58 x 10( -3) s(-1) for ATP, 8.8 x 10(-3) s(-1) for GDP, and >0.3 s(-1) for ADP at pH 6.7 and 4 degrees C, The rates were fairly pH-and temperature-de pendent. The distribution constant K-c' (=k(+1)/k(-1)) between the tig ht and loose complexes ranged between 2 and 30, suggesting formation o f 71-97% of the tight complex at equilibrium. The K-c' decreases with increasing pH, indicating a progressively less tight complex populatio n. Anions (SO42-) form a loose complex with UCP, thus affecting the in itial association step, but not the subsequent transition step. While the kinetic constants were verified by dilution and chase experiments as well as in mass action plots, they were further corroborated with d ata obtained by fluorescence competition measurements. Taken to togeth er, our results show that nucleotide binding to UCP occurs via a two-s tage mechanism in which the initial loose complex rearranges slowly in to a tight complex.