- Title
- Identification of cytochrome-b5 reductase as the enzyme responsible for NADH-dependent lucigenin chemiluminescence in human spermatozoa
- Creator
- Baker, Mark A.; Krutskikh, Anton; Curry, Benjamin J.; Hetherington, Louise; Aitken, Robert John
- Relation
- Biology of Reproduction Vol. 73, no. 2, p. 334-342
- Publisher
- Society for the Study of Reproduction
- Resource Type
- journal article
- Date
- 2005
- Description
- 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.
- Subject
- cytochrome-b5 reductase; female reproductive tract; lucigenin; reactive; oxygen species; sperm; sperm capacitation; sperm maturation; spermatozoa; WST-1; oxygen species generation; superoxide anion production; human sperm; capacitation; bis-n-methylacridinium; necrosis-factor-alpha; radical; production; tyrosine phosphorylation; tetrazolium salt; oxidase; activity; redox activity
- Identifier
- http://hdl.handle.net/1959.13/24450
- Identifier
- uon:41
- Identifier
- ISSN:1529-7268
- Language
- eng
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