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Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6'-O-fluorescein-atractyloside
- Source :
- The Biochemical journal. 376(Pt 3)
- Publication Year :
- 2003
-
Abstract
- Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6´-O-fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family.
- Subjects :
- congenital, hereditary, and neonatal diseases and abnormalities
Protein Conformation
Dimer
Biology
Mitochondrion
Atractyloside
Biochemistry
chemistry.chemical_compound
Protein structure
Animals
Fluorescein
Enzyme Inhibitors
Molecular Biology
Fluorescent Dyes
Chromatography
Biological Transport
Cell Biology
Mitochondrial carrier
Chromatography, Agarose
Chromatography, Ion Exchange
Mitochondria
Adenosine Diphosphate
Adenosine diphosphate
Spectrometry, Fluorescence
chemistry
Cattle
ATP–ADP translocase
Mitochondrial ADP, ATP Translocases
Research Article
Subjects
Details
- ISSN :
- 14708728
- Volume :
- 376
- Issue :
- Pt 3
- Database :
- OpenAIRE
- Journal :
- The Biochemical journal
- Accession number :
- edsair.doi.dedup.....89d6738de53cea45a4bf8e91ec2bde34