eTopics

eTopic 13.1 Observing Energy Carriers in Living Cells

Energy carriers lead a dynamic existence, often with rapid turnover. How are energy carriers measured in living cells? One way is to use nuclear magnetic resonance (NMR) spectroscopy (Fig. 1). The use of NMR for real-time observation of live organisms was pioneered in the 1970s by Robert Shulman and colleagues at Yale University. Further advances led to magnetic resonance imaging (MRI), a technique in which the human body is scanned for NMR signals to build a three-dimensional image. MRI is now the method of choice for noninvasive imaging of patients with tumors, stroke, or other internal defects.

NMR spectroscopy gives detailed information about molecules that contain nuclei whose atomic number or mass number is an odd number, and that therefore possess nuclear magnetic moment. Nuclei particularly useful in biochemistry include 1H, 13C, and 31P. NMR detects nuclear spin transitions associated with the nuclear magnetic moment of nuclei suspended within the intense magnetic field of a superconducting magnet. The spin transition occurs as the nuclear magnetic moment of each atom interacts with electromagnetic radiation, producing a signal.

Within live cells, NMR can be used to observe concentrations of molecules with extremely high turnover, such as ATP and NADH (Fig. 1). These energy carriers are revealed by NMR signals from their phosphate groups. The naturally occurring phosphorus isotope 31P has a nuclear spin, which generates the nuclear magnetic moment required for an NMR signal. The 31P NMR spectrum of live E. coli cells reveals several peaks corresponding to cytoplasmic levels of nucleotide triphosphates (primarily ATP) and NADH. Each phosphorus signal position is “shifted” by shielding from electrons in molecular orbitals. The shift differs for each phosphorus in the molecule.

In Figure 1, the levels of phosphorylated energy carriers were used to assess the overall energy levels of E. coli cells exposed to tellurite, a toxic chemical. The 31P NMR spectrum reveals three shift peaks corresponding to the three phosphates of ATP and the two phosphates of ADP. The middle phosphate of ATP (ATP β) generates a well-isolated peak indicating ATP concentration. As the overall energy level of the cell rises upon addition of glucose, the ATP β peak increases. Thus, the concentration of phosphorylated energy carriers serves as a measure of energy flux within the cell. Similar measurements for human tissues are used to study the biochemistry of athletic performance.

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Figure 1  Phosphorylated energy carriers observed by 31P NMR.  The 31P NMR spectrum reveals peaks corresponding to cytoplasmic levels of nucleotide triphosphates (primarily ATP) and NADH. The alpha-phosphate links directly to the nucleotide; the beta-phosphate lies between two phosphates; and the gamma-phosphate is terminal. The beta-phosphate of ATP (ATP β) generates a well-isolated peak indicating ATP concentration. Source: Elke M. Lohmeier-Vogel et al. 2004. Appl. Environ. Microbiol. 70:7342.