Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa.
Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.
Baker
Mark A
MA
The 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.
Krutskikh
Anton
A
Curry
Benjamin J
BJ
Hetherington
Louise
L
Aitken
R John
RJ
D000166:Acridines / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D004396:Coloring Agents / Q000494:pharmacology; D042966:Cytochrome-B(5) Reductase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008163:Luminescent Measurements; D008297:Male; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011719:Pyrazines / Q000494:pharmacology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence; D013094:Spermatozoa / Q000201:enzymology* / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D014162:Transfection
C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D000166:Acridines; D004396:Coloring Agents; D007093:Imidazoles; D011719:Pyrazines; D013778:Tetrazolium Salts; D009243:NAD; C063412:2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-alpha)pyrazin-3-one; C033472:10,10'-dimethyl-9,9'-biacridinium; D012313:RNA; D042966:Cytochrome-B(5) Reductase