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

Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061, USA.
Biophysical Journal (Impact Factor: 3.97). 05/2001; 80(4):2018-28. DOI: 10.1016/S0006-3495(01)76172-3
Source: PubMed


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 (Q(10) 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.

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    • "With these considerations, toxicity and bleaching can be avoided over recording periods of hours, as measured from the reproducibility of synaptically-evoked responses. Alternatively, deliberate bleaching of NADH with very high intensity UV flashes can be useful for kinetic studies of tissue NADH generation, as the enzymatic regeneration of NADH within tissues can be monitored (Combs and Balaban, 2001, 2004; Joubert et al., 2004) From early studies of a range of tissues, it was concluded that the intensity of cellular NADH fluorescence was not uniform, but was strongly increased in the mitochondrial compartment, and quenched when located in the cytosol (Chance and Baltscheffsky, 1958).The reason(s) for enhanced mitochondrial fluorescence was not immediately established, but association with matrix proteins was considered a major contributor (Chance and Baltscheffsky, 1958; Avi-Dor et al., 1962; Estabrook, 1962). Jobsis and colleagues concluded that NADH fluorescence measured in intact cat cortex was primarily mitochondrial, and that enhancement of fluorescence was due to mitochondrial dehydrogenases (Jobsis et al., 1971). "
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