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Biophys J, April 2001, p. 2018-2028, Vol. 80, No. 4

Direct Imaging of Dehydrogenase Activity within Living Cells Using Enzyme-Dependent Fluorescence Recovery after Photobleaching (ED-FRAP)

C. A. Combs and R. S. Balaban

Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061 USA

Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q10 of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD+) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.

Biophys J, April 2001, p. 2018-2028, Vol. 80, No. 4
© 2001 by the Biophysical Society   0006-3495/01/04/2018/11  $2.00


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