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J. Biol. Chem., Vol. 280, Issue 26, 99932, July 1, 2005

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Global Analysis Reveals Concentrations of Free and Enzyme-bound NADH

Nicotinamide adenine dinucleotide (NADH) is the principal electron donor in metabolism and thus one of the most important coenzymes in the cell. The fact that the molecule is a natural fluorophore makes it an ideal non-invasive fluorescent probe of metabolic state. However, quantitating free and enzyme-bound NADH within cells has not been easy. Current analytical chemistry techniques generally entail destroying the tissue and limiting their use to single-shot measurements, and fluorescent techniques cannot distinguish between free and enzyme-bound NADH.

Hippocampal tissue fluorescence during base line (A) is enhanced after a brief episode of hypoxia (B).

In this Paper of the Week, Harshad D. Vishwasrao and colleagues report that a combined analysis of time-resolved fluorescence and anisotropy decays allowed them to follow changes in amounts of free and enzyme-bound NADH in living tissue. In particular, binding of NADH to an enzyme greatly increases the rotation time of the bound fluorophore, so anisotropy measurements give a sensitive approach to quantitation of free and enzyme-bound NADH in living cells. Importantly, the authors demonstrated that a substantial fraction of NADH in the tissue analyzed is free, in contrast to earlier reports, which maintained that all NADH is enzyme-bound. Also, a metabolic transition to hypoxia changes the population of bound NADH molecules, as reflected in the distribution of species with different lifetimes.

FOOTNOTES

See referenced article, J. Biol. Chem. 2005, 280, 25119-25126

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Search for Related Content Related Collections Papers Of The Week