Identification of Cytochrome P450-rRductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations

Mark A. Baker, Anton Krutskikh, Benjamin J. Curry, Eileen A. McLaughlin, R. John Aitken

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Lucigenin-dependent chemiluminescence and WST-1 reduction can be detected following addition of NADPH to many cell types, including rat epididymal sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other probes-such as MCLA and luminol-that are capable of detecting reactive oxygen metabolites do not produce a chemiluminescent signal in this model system. Our aim was to purify and identify the enzyme catalyzing the NADPH-dependent lucigenin and WST-1 reduction from rat epididymal spermatozoa preparations. Here, we show the identity of this enzyme as cytochrome P450-reductase. In support of this, a homogenous preparation of this protein was capable of reducing lucigenin and WST-1 in the presence of NADPH. Moreover, COS-7 cells overexpressing cytochrome P450-reductase displayed a 3-fold increase in the aforementioned activity compared with mock-transfected cells. Immunolocalization studies and biochemical analysis suggest that the majority of the NADPH-lucigenin activity is localized to the epithelial cells present within the epididymis. These results emphasize the importance of the direct NADPH-dependent reduction of superoxide-sensitive probes by cytochrome P450-reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Original languageEnglish
Pages (from-to)307-318
Number of pages12
JournalBiology of Reproduction
Volume71
Issue number1
DOIs
Publication statusPublished - 1 Jul 2004
Externally publishedYes

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Tetrazolium Salts
NADP
Cytochrome P-450 Enzyme System
Spermatozoa
NADPH-Ferrihemoprotein Reductase
Enzymes
Reactive Oxygen Species
Luminol
Epididymis
COS Cells
Luminescence
Superoxides
Suspensions
Epithelial Cells
10,10'-dimethyl-9,9'-biacridinium
Oxygen
Proteins

Cite this

Baker, Mark A. ; Krutskikh, Anton ; Curry, Benjamin J. ; McLaughlin, Eileen A. ; Aitken, R. John. / Identification of Cytochrome P450-rRductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations. In: Biology of Reproduction. 2004 ; Vol. 71, No. 1. pp. 307-318.
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Identification of Cytochrome P450-rRductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations. / Baker, Mark A.; Krutskikh, Anton; Curry, Benjamin J.; McLaughlin, Eileen A.; Aitken, R. John.

In: Biology of Reproduction, Vol. 71, No. 1, 01.07.2004, p. 307-318.

Research output: Contribution to journalArticle

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