Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.

TitleNucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.
Publication TypeJournal Article
Year of Publication2002
AuthorsJekabsons, MB, Echtay, KS, Brand, MD
JournalBiochem J
IssuePt 2
Date Published2002 Sep 01
KeywordsAdenosine Triphosphate, Chromatography, Cloning, Molecular, Detergents, Durapatite, Humans, Inclusion Bodies, Ion Channels, Kidney, Kinetics, Membrane Transport Proteins, Mitochondria, Mitochondrial Proteins, Protein Folding, Proteins, Recombinant Proteins, Regression Analysis, Uncoupling Agents, Uncoupling Protein 2

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.

Alternate JournalBiochem. J.
Citation Key10.1042/BJ20020469
PubMed ID12030845
PubMed Central IDPMC1222799