Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6'-O-fluorescein-atractyloside.

TitleAltered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6'-O-fluorescein-atractyloside.
Publication TypeJournal Article
Year of Publication2003
AuthorsSmith, VR, Fearnley, IM, Walker, JE
JournalBiochem J
Volume376
IssuePt 3
Pagination757-63
Date Published2003 Dec 15
ISSN1470-8728
KeywordsAdenosine Diphosphate, Animals, Atractyloside, Biological Transport, Cattle, Chromatography, Agarose, Chromatography, Ion Exchange, Enzyme Inhibitors, Fluorescein, Fluorescent Dyes, Mitochondria, Mitochondrial ADP, ATP Translocases, Protein Conformation, Spectrometry, Fluorescence
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.

DOI10.1042/BJ20030942
Alternate JournalBiochem. J.
Citation Key10.1042/BJ20030942
PubMed ID14498831
PubMed Central IDPMC1223817