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Combs 2004 Methods Enzymol

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Publications in the MiPMap
Combs CA, Balaban RS (2004) Enzyme-dependent fluorescence recovery after photobleaching of NADH: in vivo and in vitro applications to the study of enzyme kinetics. Methods Enzymol 385:257-86.

Β» PMID: 15130744

Combs CA, Balaban RS (2004) Methods Enzymol

Abstract: This chapter provides a brief overview of the use of nicotinamide adenine dinucleotide (NADH) fluorescence in biochemistry and biology and the photophysics of NADH. Enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) has been described as a method of monitoring enzymatic activities in microsamples in vitro or cells using confocal microscopy, as well as in larger samples of enzyme solutions or organelle suspensions. The use of NADH ED-FRAP to evaluate the turnover of NADH in vitro and in vivo is described with a focus on experimental details. Examples of performing NADH ED-FRAP experiments on isolated enzymes, mitochondria, and cells are outlined. It is found that absolute quantification of the total pyridine nucleotide pool from tissue is dependent on the assay method. NADH ED-FRAP has been shown to be effective in measuring dehydrogenase activity in several enzyme systems in vitro as well as suspensions of isolated mitochondria. The NADH ED-FRAP measurement of kinetic parameters in isolated mitochondria is also elaborated.

β€’ Bioblast editor: Doerrier C


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