Faculty Opinions recommendation of Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.

Author(s):  
Daniel Ricquier
2002 ◽  
Vol 366 (2) ◽  
pp. 565-571 ◽  
Author(s):  
Mika B. JEKABSONS ◽  
Karim S. ECHTAY ◽  
Martin D. BRAND

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.


Author(s):  
Wafaa A. Shehata ◽  
Mostafa A. Hammam ◽  
Rasha S. Enany ◽  
Sally M. El‐Hefnawy ◽  
Shimaa Abdelsattar

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Ji Hye Lim ◽  
Mi Mi Ko ◽  
Hoyoung Lee ◽  
Ho Yeon Go ◽  
Tae-Woong Moon ◽  
...  

Uncoupling protein 2 (UCP2), a mitochondrial protein present in many organs and cell types, is known to dissipate the proton gradient formed by the electron transport chain. Its function is correlated with predictive parameters, such as obesity, diabetes, and metabolic syndromes. We analyzed the distribution of UCP2 polymorphisms in stroke patients diagnosed with one of the following four stroke subtypes based on the TKM standard pattern identification (PI): Qi-deficiency (QD), Dampness and Phlegm (D&P), Yin-deficiency (YD), and Fire and Heat (F&D). We studied a total of 1,786 stroke patients (397/QD, 645/D&P, 223/YD, and 522/F&D, 586/normal). Genotyping for the G-1957A, G-866A and A55V UCP2 polymorphisms was performed using the TaqMan. G-866A and A55V were significantly associated with the D&P and H&F subtypes. The frequency of subjects with the A allele of G-866A was significantly lower than the frequency of subjects with the GG type. The A55V polymorphism was also shown similar effect with G-866A in the dominant model. In contrast, no SNPs were shown to be associated with the QD or YD subtypes in this study. These results showed that the G-866A and A55V UCP2 polymorphisms may be genetic factors for specific PI types among Korean stroke patients.


Life Sciences ◽  
1998 ◽  
Vol 64 (3) ◽  
pp. PL41-PL50 ◽  
Author(s):  
N. Tu ◽  
H. Chen ◽  
U. Winnikes ◽  
I. Reinert ◽  
G. Marmann ◽  
...  

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