Rapid fluorescent visualization of multiple NAD(P)H oxidoreductases in homogenate, permeabilized cells, and tissue slices.
Anal Biochem
; 440(2): 189-96, 2013 Sep 15.
Article
em En
| MEDLINE
| ID: mdl-23747529
ABSTRACT
Intracellular NAD(P)H oxidoreductases are a class of diverse enzymes that are the key players in a number of vital processes. The method we present and validate here is based on the ability of many NAD(P)H oxidoreductases to reduce the superoxide probe lucigenin, which is structurally similar to flavins, to its highly fluorescent water-insoluble derivative dimethylbiacridene. Two modifications of the method are proposed (i) an express method for tissue homogenate and permeabilized cells in suspensions and (ii) a standard procedure for cells in culture and acute thin tissue slices. The method allows one to assess, visualize, and localize, using fluorescent markers of cellular compartments, multiple NADH and NADPH oxidoreductase activities. The application of selective inhibitors (e.g., VAS2870, a NOX2 inhibitor; plumbagin, a NOX4 inhibitor) allows one to distinguish and compare specific NAD(P)H oxidoreductase activities in cells and tissues and to attribute them to known enzymes. The method is simple, rapid, and flexible. It can be easily adapted to a variety of tasks. It will be useful for investigations of the role of various NAD(P)H oxidoreductases in a number of physiological and pathophysiological processes.
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Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Imagem Molecular
/
NADH NADPH Oxirredutases
Limite:
Animals
Idioma:
En
Revista:
Anal Biochem
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Federação Russa