Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues
Introduction
Mitochondria are the major cellular source of adenosine triphosphate (ATP) synthesis. Its oxidative phosphorylation system generates 36 molecules of ATP per molecule of glucose, as opposed to only 2 molecules of ATP generated by glycolysis in the cytoplasm. Therefore, the measurement of mitochondrial ATP synthesis can be considered a pivotal tool in understanding many important characteristics of cellular energy metabolism, both in the normal physiological state and in pathological conditions such as mitochondrial disorders. In the first section of this chapter, we will discuss approaches to measure ATP synthesis from mammalian cells and tissues. In the second section, we will discuss procedures to estimate the steady‐state content of ATP and other high‐energy phosphates by high‐performance liquid chromatography (HPLC).
Section snippets
ATP Synthesis Assays
Two methodological issues need to be addressed when measuring mitochondrial ATP synthesis. The first one is how to deliver reaction substrates to the mitochondria. Because of the low permeability of the plasma membrane to some hydrophilic substrates, such as adenosine diphosphate (ADP), some investigators prefer to analyze isolated mitochondria (Tatuch 1993, Tuena de Gomez‐Puyou 1984, Vazquez‐Memije 1996). However, for ADP phosphorylation to take place, mitochondria need to be maintained intact
Cell Permeabilization (Detergent Titration in Cultured Cells)
Although more convenient than isolating coupled mitochondria, permeabilization procedures also require standardization. Insufficient permeabilization could result in an underestimation of ATP synthesis due to lack of available substrates. Conversely, excessive permeabilization leads to mitochondrial membrane damage and uncoupling. For these reasons, it is important to establish the optimal amount of detergent needed per unit of cell protein. We use digitonin as the detergent of choice because
Experimental Procedures
This section describes the assay of ATP synthesis in five cell types: HeLa, COS‐7, N2A, HEK 293T, and 143B‐derived cytoplasmic hybrids (cybrids; King and Attardi, 1989) harboring either wild‐type mitochondrial DNA (mtDNA) or the T8993G mtDNA mutation in the ATPase 6 gene, which is responsible for a mitochondrial disorder characterized by neuropathy, ataxia, and retinitis pigmentosa (NARP; Holt et al., 1990).
Measurement of High‐Energy Phosphates in Animal Tissue and Cultured Cells by HPLC
Among the different methods used to assay high‐energy phosphates in biological samples, HPLC has the advantage of high sensitivity and efficiency because it allows for the simultaneous analyses of all species of phosphorylated nucleotides in one analysis.
Different HPLC techniques are commonly used to separate nucleotides (Zakaria and Brown, 1981). Nucleotide phosphates are charged molecules that can be separated well by ion‐exchange HPLC. However, the separation time is relatively long, and the
Acknowledgments
This work was supported by NIH grant K02‐NS47306 and by the Muscular Dystrophy Association.
References (35)
- et al.
Application and validation of an ion‐exchange high‐performance liquid chromatographic method for measuring adenine nucleotides, creatine and creatine phosphate in mouse brain
J. Chromatogr.
(1990) - et al.
Effects of chronic dietary creatine feeding on cardiac energy metabolism and on creatine content in heart, skeletal muscle, brain, liver and kidney
J. Mol. Cell. Cardiol.
(1998) - et al.
Altered properties of mitochondrial ATP‐synthase in patients with a T→G mutation in the ATPase 6 (subunit a) gene at position 8993 of mtDNA
Biochim. Biophys. Acta
(1995) - et al.
Functional characterization of mitochondrial oxidative phosphorylation in saponin‐skinned human muscle fibers
Biochim. Biophys. Acta
(1993) - et al.
Adenosine triphosphate: Continuous measurement in mitochondrial suspension by firefly luciferase luminescence
Biochem. Biophys. Res. Commun.
(1973) - et al.
Continuous monitoring of ATP levels in living insulin secreting cells expressing cytosolic firefly luciferase
FEBS Lett.
(1998) - et al.
Maintenance of regional chemical integrity for energy metabolites in microwave heat inactivated mouse brain
Brain Res. Bull.
(1984) - et al.
Optimized analysis of intracellular adenosine and guanosine phosphates in Escherichia coli
Anal. Biochem.
(1999) - et al.
Quantitating adenylate nucleotides in diverse organisms
J. Biochem. Biophys. Methods
(2005) - et al.
Focused microwave irradiation of the brain preserves in vivo protein phosphorylation: Comparison with other methods of sacrifice and analysis of multiple phosphoproteins
J. Neurosci. Methods
(2004)