Chapter 15
Determination of Adenine Nucleotide Concentrations
in Cells and Tissues by High-Performance Liquid
Chromatography
Noemı ́Garcı ́a-Tardo ́n and Bruno Guigas
Abstract
The serine/threonine AMP-activated protein kinase (AMPK) is a central player in the regulation of energy
homeostasis, and its activity is tightly controlled, among other mechanisms, by subtle changes in cellular
adenine nucleotide levels. In this chapter, we describe a step-by-step protocol for rapid, highly sensitive,
reproducible, and simultaneous determination of ATP, ADP, and AMP concentrations in cell or tissue
samples by reversed-phase high-performance liquid chromatography (HPLC).
Key wordsHPLC, Adenine nucleotides, Tissue, Cells, Quantification, Adenylate energy charge1 Introduction
The evolutionary-conserved and ubiquitously expressed serine/
threonine AMP-activated protein kinase (AMPK) is a central player
in the regulation of energy homeostasis [1]. AMPK is a heterotri-
meric protein complex, consisting of a catalyticαsubunit and two
regulatoryβandγsubunits, that functions as an adenylate charge-
regulated kinase which constantly senses the cellular energy status
by monitoring intracellular AMP, ADP, and ATP levels [2]. In
response to metabolic stresses and cellular ATP depletion, a con-
comitant rise in AMP occurs due to the presence of adenylate
kinase, leading to allosteric activation of AMPK by direct binding
to the cystathionineβ-synthase (CBS) domains onγsubunit. In
addition, the interaction between AMP and the CBS domains also
promotes phosphorylation of the Thr172 residue within the activa-
tion loop of theα-catalytic subunit by upstream AMPK kinases
(AMPKK), considerably amplifying AMPK activity. ADP has also
been shown to bind instead of ATP to some of the CBS nucleotide-
binding sites and to enhance AMPKK-mediated Thr172 phosphor-
ylation [3]. AMP and ADP also sustain AMPK activation throughDietbert Neumann and Benoit Viollet (eds.),AMPK:MethodsandProtocols, Methods in Molecular Biology, vol. 1732,
https://doi.org/10.1007/978-1-4939-7598-3_15,©Springer Science+Business Media, LLC 2018
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