Sg. Huang et al., SLOW-PHASE KINETICS OF NUCLEOTIDE-BINDING TO THE UNCOUPLING PROTEIN FROM BROWN ADIPOSE-TISSUE MITOCHONDRIA, The Journal of biological chemistry, 273(2), 1998, pp. 859-864
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.