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 3–5μM 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 2–5μM 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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September 2002
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Research Article|
September 01 2002
Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies
Mika B. JEKABSONS;
Mika B. JEKABSONS
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, U.K.
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Karim S. ECHTAY;
Karim S. ECHTAY
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, U.K.
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Martin D. BRAND
Martin D. BRAND
1
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, U.K.
1To whom correspondence should be addressed (e-mail martin.brand@mrc-dunn.cam.ac.uk).
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Publisher: Portland Press Ltd
Received:
March 25 2002
Revision Received:
May 13 2002
Accepted:
May 27 2002
Accepted Manuscript online:
May 27 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2002
2002
Biochem J (2002) 366 (2): 565–571.
Article history
Received:
March 25 2002
Revision Received:
May 13 2002
Accepted:
May 27 2002
Accepted Manuscript online:
May 27 2002
Citation
Mika B. JEKABSONS, Karim S. ECHTAY, Martin D. BRAND; Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies. Biochem J 1 September 2002; 366 (2): 565–571. doi: https://doi.org/10.1042/bj20020469
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