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) from brown adipose tissue mitochondria were studied with a filter binding method. Fast and slow phases of binding were observed, corresponding to the two-stage binding model based on equilibrium binding studies (Huang, S. G., and Klingenberg, M. (1996) Biochemistry 35, 7846-7854) (Reaction 1). [reaction: see text] Although this method determines total binding, only the slow phase can be resolved. The fast unresolved phase represents the formation of the initial loose UCP-nucleotide complex (UN; Kd approximately 2 microM), whereas the slow phase reflects the tight binding (U*N) associated with a conformational change induced by 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 dissociation 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-dependent. The distribution constant Kc' (=k+1/k-1) between the tight and loose complexes ranged between 2 and 30, suggesting formation of 71-97% of the tight complex at equilibrium. The Kc' decreases with increasing pH, indicating a progressively less tight complex population. Anions (SO42-) form a loose complex with UCP, thus affecting the initial 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 data obtained by fluorescence competition measurements. Taken together, our results show that nucleotide binding to UCP occurs via a two-stage mechanism in which the initial loose complex rearranges slowly into a tight complex.
Huang
S G
SG
Institute of Physical Biochemistry, University of Munich, Schillerstrasse 44, D-80336 Munich, Federal Republic of Germany. Sghuang@pbm.med.uni-muenchen.de
Lin
Q S
QS
Klingenberg
M
M
D000227:Adenine Nucleotides / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D005374:Filtration; D006150:Guanine Nucleotides / Q000378:metabolism*; D007473:Ion Channels; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D000071256:Uncoupling Protein 1
D000227:Adenine Nucleotides; D002352:Carrier Proteins; D006150:Guanine Nucleotides; D007473:Ion Channels; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D000071256:Uncoupling Protein 1