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Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations

Journal Article


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.

Publication Date


  • 2004

Citation


  • Baker, M. A., Krutskikh, A., Curry, B. J., McLaughlin, E. A., & Aitken, R. J. (2004). Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biology of Reproduction, 71(1), 307-318. doi:10.1095/biolreprod.104.027748

Scopus Eid


  • 2-s2.0-3142748299

Start Page


  • 307

End Page


  • 318

Volume


  • 71

Issue


  • 1

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.

Publication Date


  • 2004

Citation


  • Baker, M. A., Krutskikh, A., Curry, B. J., McLaughlin, E. A., & Aitken, R. J. (2004). Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations. Biology of Reproduction, 71(1), 307-318. doi:10.1095/biolreprod.104.027748

Scopus Eid


  • 2-s2.0-3142748299

Start Page


  • 307

End Page


  • 318

Volume


  • 71

Issue


  • 1