Time-resolved ATP measurements during vesicle respiration

Jing Lin, Dominik Weixler, Sven Daboss, Gerd M. Seibold*, Corina Andronescu, Wolfgang Schuhmann, Christine Kranz

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ± 0.03 μM/μl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ± 0.02 μM/μl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved.

Original languageEnglish
Article number120083
JournalTalanta
Volume205
Number of pages7
ISSN0039-9140
DOIs
Publication statusPublished - 2019

Keywords

  • ATP microbiosensor
  • Fluorescence microscopy
  • Gram negative cells
  • Inhibition
  • Inverted vesicles
  • Scanning electrochemical microscopy

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