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Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules

Biological Procedures Online volume 10, pages 36–46 (2008)Cite this article

Abstract

Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.

Abbreviations

2D-TLC:

two-dimensional thin layer chromatography

acetyl-P:

acetyl phosphate

PTA:

phosphotransacetylase

PDHC:

pyruvate dehydrogenase complex

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Authors and Affiliations

  1. Department of Microbiology and Immunology Loyola University Chicago, 2160 S. First Ave. Bldg. 105, 60153, Maywood, IL, USA

    David H. Keating, Ana Shulla, Adam H. Klein & Alan J. Wolfe

Authors
  1. David H. Keating
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  2. Ana Shulla
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  3. Adam H. Klein
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  4. Alan J. Wolfe
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Correspondence to Alan J. Wolfe.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Keating, D.H., Shulla, A., Klein, A.H. et al. Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules. Biol. Proced. Online 10, 36–46 (2008). https://doi.org/10.1251/bpo141

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  • DOI: https://doi.org/10.1251/bpo141

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Biological Procedures Online

ISSN: 1480-9222

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