Abstract
Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90% pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, N-methylanthraniloyl-tagged purine nucleoside di- and tri-phosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by least-squares non-linear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 3-5 microM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with [8-14C]ATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 2-5 microM affinity. Affinities for ADP and GDP were approx. 10-fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosyl-solubilized bacterial inclusion bodies by a two-step treatment with C12E9 detergent and hydroxyapatite; (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity; (c) binds GDP with the same affinity as GDP inhibits superoxide-stimulated uncoupling by kidney mitochondria; and (d) exhibits a different nucleotide preference than kidney mitochondria.
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Selected References
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- Echtay K. S., Winkler E., Frischmuth K., Klingenberg M. Uncoupling proteins 2 and 3 are highly active H(+) transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone). Proc Natl Acad Sci U S A. 2001 Feb 13;98(4):1416–1421. doi: 10.1073/pnas.98.4.1416. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Echtay Karim S., Roussel Damien, St-Pierre Julie, Jekabsons Mika B., Cadenas Susana, Stuart Jeff A., Harper James A., Roebuck Stephen J., Morrison Alastair, Pickering Susan. Superoxide activates mitochondrial uncoupling proteins. Nature. 2002 Jan 3;415(6867):96–99. doi: 10.1038/415096a. [DOI] [PubMed] [Google Scholar]
- Flatmark T., Pedersen J. I. Brown adipose tissue mitochondria. Biochim Biophys Acta. 1975 Mar 31;416(1):53–103. doi: 10.1016/0304-4173(75)90013-0. [DOI] [PubMed] [Google Scholar]
- Fleury C., Neverova M., Collins S., Raimbault S., Champigny O., Levi-Meyrueis C., Bouillaud F., Seldin M. F., Surwit R. S., Ricquier D. Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet. 1997 Mar;15(3):269–272. doi: 10.1038/ng0397-269. [DOI] [PubMed] [Google Scholar]
- Heaton G. M., Nicholls D. G. The structural specificity of the nucleotide-binding site and the reversible nature of the inhibition of proton conductance induced by bound nucleotides in brown-adipose-tissue mitochondria. Biochem Soc Trans. 1977;5(1):210–212. doi: 10.1042/bst0050210. [DOI] [PubMed] [Google Scholar]
- Huang S. G., Klingenberg M. Fluorescent nucleotide derivatives as specific probes for the uncoupling protein: thermodynamics and kinetics of binding and the control by pH. Biochemistry. 1995 Jan 10;34(1):349–360. doi: 10.1021/bi00001a043. [DOI] [PubMed] [Google Scholar]
- Jabůrek M., Varecha M., Gimeno R. E., Dembski M., Jezek P., Zhang M., Burn P., Tartaglia L. A., Garlid K. D. Transport function and regulation of mitochondrial uncoupling proteins 2 and 3. J Biol Chem. 1999 Sep 10;274(37):26003–26007. doi: 10.1074/jbc.274.37.26003. [DOI] [PubMed] [Google Scholar]
- Klingenberg M. Nucleotide binding to uncoupling protein. Mechanism of control by protonation. Biochemistry. 1988 Jan 26;27(2):781–791. doi: 10.1021/bi00402a044. [DOI] [PubMed] [Google Scholar]
- Lin C. S., Klingenberg M. Characteristics of the isolated purine nucleotide binding protein from brown fat mitochondria. Biochemistry. 1982 Jun 8;21(12):2950–2956. doi: 10.1021/bi00541a023. [DOI] [PubMed] [Google Scholar]
- Lin C. S., Klingenberg M. Isolation of the uncoupling protein from brown adipose tissue mitochondria. FEBS Lett. 1980 May 5;113(2):299–303. doi: 10.1016/0014-5793(80)80613-2. [DOI] [PubMed] [Google Scholar]
- Nicholls D. G. Brown adipose tissue mitochondria. Biochim Biophys Acta. 1979 Jul 3;549(1):1–29. doi: 10.1016/0304-4173(79)90016-8. [DOI] [PubMed] [Google Scholar]
- Pecqueur C., Alves-Guerra M. C., Gelly C., Levi-Meyrueis C., Couplan E., Collins S., Ricquier D., Bouillaud F., Miroux B. Uncoupling protein 2, in vivo distribution, induction upon oxidative stress, and evidence for translational regulation. J Biol Chem. 2000 Nov 29;276(12):8705–8712. doi: 10.1074/jbc.M006938200. [DOI] [PubMed] [Google Scholar]
- Pedersen J. I. Coupled endogenous respiration in brown adipose tissue mitochondria. Eur J Biochem. 1970 Sep;16(1):12–18. doi: 10.1111/j.1432-1033.1970.tb01047.x. [DOI] [PubMed] [Google Scholar]
- Rial E., González-Barroso M., Fleury C., Iturrizaga S., Sanchis D., Jiménez-Jiménez J., Ricquier D., Goubern M., Bouillaud F. Retinoids activate proton transport by the uncoupling proteins UCP1 and UCP2. EMBO J. 1999 Nov 1;18(21):5827–5833. doi: 10.1093/emboj/18.21.5827. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schroers A., Burkovski A., Wohlrab H., Krämer R. The phosphate carrier from yeast mitochondria. Dimerization is a prerequisite for function. J Biol Chem. 1998 Jun 5;273(23):14269–14276. doi: 10.1074/jbc.273.23.14269. [DOI] [PubMed] [Google Scholar]
- Stuart J. A., Harper J. A., Brindle K. M., Jekabsons M. B., Brand M. D. Physiological levels of mammalian uncoupling protein 2 do not uncouple yeast mitochondria. J Biol Chem. 2001 Feb 22;276(21):18633–18639. doi: 10.1074/jbc.M011566200. [DOI] [PubMed] [Google Scholar]