Slow-phase Kinetics of Nucleotide Binding to the Uncoupling Protein from Brown Adipose Tissue Mitochondria

Abstract

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). ؊1 for ADP at pH 6.7 and 4°C. The rates were fairly pH-and temperature-dependent. The distribution constant K c (‫؍‬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 K c decreases with increasing pH, indicating a progressively less tight complex population. Anions (SO 4 2؊ ) 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.

Activating ω-6 Polyunsaturated Fatty Acids and Inhibitory Purine Nucleotides Are High Affinity Ligands for Novel Mitochondrial Uncoupling Proteins UCP2 and UCP3

“…We must admit that sulfate used in our assay media strongly decreases the PN affinity to UCP1 (25) and presumably also to UCP2/UCP3, which might explain the discrepancies between our K i values and those measured by Klingenberg and co-workers (21,58) and partly also between the K i values (Table II) (Table III). We chose sulfate to ensure that the anion used would be membrane impermeant, would not quench the SPQ probe, and that UCPs would not transport it.…”

“…the lowering of PN inhibition by some yet unknown factors, may represent the mechanism that activates UCP2-or UCP3-mediated uncoupling in vivo. Such well known modulators for UCP1 are alkaline pH and Mg 2ϩ (24,25). Also, cofactors like coenzyme Q 10 (21,26) and superoxide (27) were suggested to activate UCP2 (UCP3).…”

Temperature dependence of ligand–protein complex formation as reflected by saturation transfer difference NMR experiments

“…The lactose-binding properties of growth-regulatory galectin7 (p53-induced gene 1), 5,6 as well as the specificity of nucleotides for human annexin A6, 7 and the uncoupling protein 1 (UCP1) from brown adipose tissue mitochondria, 8 are well defined. In contrast, the characterization of downstream regulatory element antagonist modulator of the human prodynorphin gene (DREAM) binding to tryptophan-containing peptides is an example of a work in progress in which few binding or kinetic parameters are known.…”

“…Galectin7, 5 annexin A6, 7 UCP-1, 8 and RNase S 13 were expressed and purified according to established protocols. His-tagged DREAM (residues 71-256) was expressed and purified according to the manufacturers' protocols (Invitrogen/BD Biosciences) with characterization (DNA sequencing of inserted gene, polyacrylamide gel analysis, Western blot, and mass spectroscopy) carried out in-house.…”

“…8 Yet the trend may suggest a value of approximately 1 s 1 at 310 K (as 10 K increases the off-rate by ca a factor of 10) and, therefore, a chance of observing STD effects. Unfortunately, UCP1 is unstable at higher temperatures, 14 illustrating that raised temperatures cannot always provide better STD data, and we were not able to observe any STD effects for the binding of ATP to this protein.…”