Slow-phase kinetics of nucleotide binding to the uncoupling protein from brown adipose tissue mitochondria.

摘要: Abstract The kinetics of nucleotide binding to the uncoupling protein (UCP) from brown adipose tissue mitochondria were studied with a filter method. Fast and slow phases observed, corresponding two-stage model based on equilibrium studies (Huang, S. G., Klingenberg, M. (1996) Biochemistry 35, 7846–7854) (Reaction 1). Although this method determines total binding, only phase can be resolved. fast unresolved represents formation initial loose UCP-nucleotide complex (UN;K d ≈ 2 μm), whereas reflects tight (U*N) associated conformational change induced by bound nucleotide. Best fits data yielded, for phase, k +1 values 3.0 × 10−3 s−1 GTP, 4.8 ATP, 0.13 GDP, >0.7 ADP dissociation rate constants (k −1) 0.10 10−3s−1 0.58 8.8 >0.3 at pH 6.7 4 °C. rates fairly pH- temperature-dependent. distribution constantK c′ (=k +1/k between complexes ranged 30, suggesting 71–97% equilibrium. TheK decreases increasing pH, indicating progressively less population. Anions (SO4 2−) form UCP, thus affecting association step, but not subsequent transition step. While kinetic verified dilution chase experiments as well in mass action plots, they further corroborated obtained fluorescence competition measurements. Taken together, our results show that UCP occurs via mechanism which rearranges slowly into complex.