title: Identification of cytochrome P450-reductase as the enzyme responsible for NADPH-dependent lucigenin and tetrazolium salt reduction in rat epididymal sperm preparations.
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.
authors.affiliation: ARC Centre of Excellence in Biotechnology and Development, Reproductive Science Group, School of Environmental and Life Sciences, and Hunter Medical Research Institute, University of Newcastle, Callaghan, New South Wales 2308, Australia.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Baker
authors.forename: Anton
authors.initials: A
authors.lastname: Krutskikh
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Curry
authors.forename: Eileen A
authors.initials: EA
authors.lastname: McLaughlin
authors.forename: R John
authors.initials: RJ
authors.lastname: Aitken
mesh_terms: D000166:Acridines / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004822:Epididymis; D008163:Luminescent Measurements; D008297:Male; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology / Q000502:physiology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D013094:Spermatozoa / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000302:isolation & purification / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D000166:Acridines; D013778:Tetrazolium Salts; C033472:10,10'-dimethyl-9,9'-biacridinium; D009249:NADP; D009251:NADPH-Ferrihemoprotein Reductase