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  • Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding.

Evidence for major structural changes in subunit C of the vacuolar ATPase due to nucleotide binding.

FEBS letters (2005-03-29)
Andrea Armbrüster, Christina Hohn, Anne Hermesdorf, Karin Schumacher, Michael Börsch, Gerhard Grüber
ABSTRACT

The ability of subunit C of eukaryotic V-ATPases to bind ADP and ATP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the photoaffinity and the FCS data indicate that the ATP-analogues bind more weakly to subunit C than the ADP-analogues. Site-directed mutagenesis and N-terminal sequencing of subunit C from Arabidopsis (VHA-C) and yeast (Vma5p) have been used to map the C-terminal region of subunit C as the nucleotide-binding site. Tryptophan fluorescence quenching and decreased susceptibility to tryptic digestion of subunit C after binding of different nucleotides provides evidence for structural changes in this subunit caused by nucleotide-binding.

MATERIALS
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Brand
Product Description

Sigma-Aldrich
Atto 532 maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling to thiols)
Sigma-Aldrich
Atto 532 NHS ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Sigma-Aldrich
Atto 532, BioReagent, suitable for fluorescence, ≥90% (HPCE)