Assay of Mitochondrial ATP Synthesis in Animal Cells and Tissues

https://doi.org/10.1016/S0091-679X(06)80007-5Get rights and content

Publisher Summary

This chapter discusses approaches to measure adenosine triphosphate (ATP) synthesis from mammalian cells and tissues and presents the procedures to estimate the steady-state content of ATP and other high-energy phosphates by high-performance liquid chromatography (HPLC). The isolation of highly coupled mitochondria from cultured cells involves delicate and time-consuming procedures; the results are sometimes inconsistent, leading to a potential lack of reproducibility. Measurement of ATP synthesis on whole cells requires a permeabilization step to allow for the penetration of hydrophilic substrates through biological membranes. In addition, the use of permeabilized cells rather than isolated mitochondria substantially reduces the number of cells required for each assay. Fluorimetry is a commonly used method to detect ATP produced by isolated mitochondria. Another method employed on isolated mitochondria is the incorporation of Pi into ADP and its subsequent transfer to glucose-6-phosphate by hexokinase, followed by extraction of unincorporated Pi and measurement of radioactivity in a scintillation counter. 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. The chapter also describes the assay of ATP synthesis in five cell types—HeLa, COS-7, N2A, HEK 293T, and 143 B-derived cytoplasmic hybrids 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.

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.

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