Identification of Cytochrome-b5 Reductase as the Enzyme Responsible for NADH-Dependent Lucigenin Chemiluminescence in Human Spermatozoa1
Open Access
- 1 August 2005
- journal article
- Published by Oxford University Press (OUP) in Biology of Reproduction
- Vol. 73 (2), 334-342
- https://doi.org/10.1095/biolreprod.104.037960
Abstract
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.Keywords
This publication has 46 references indexed in Scilit:
- Redox activity associated with the maturation and capacitation of mammalian spermatozoaFree Radical Biology & Medicine, 2004
- Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitationJournal of Cell Science, 2004
- Development of the signalling pathways associated with sperm capacitation during epididymal maturationMolecular Reproduction and Development, 2003
- High-Capacity Redox Control at the Plasma Membrane of Mammalian Cells: Trans-Membrane, Cell Surface, and Serum NADH-OxidasesAntioxidants and Redox Signaling, 2000
- Biochemical Characterization of Lucigenin (Bis-N-methylacridinium) as a Chemiluminescent Probe for Detecting Intramitochondrial Superoxide Anion Radical ProductionBiochemical and Biophysical Research Communications, 1999
- Superoxide anion production by human spermatozoa as a part of the ionophore‐induced acrosome reaction processInternational Journal of Andrology, 1995
- Reactive oxygen species and human spermatozoa: Analysis of the cellular mechanisms involved in luminol‐ and lucigenin‐dependent chemiluminescenceJournal of Cellular Physiology, 1992
- Isolation of motile spermatozoa by density gradient centrifugation in Percoll®Gamete Research, 1983
- Biological Defense Mechanisms. THE PRODUCTION BY LEUKOCYTES OF SUPEROXIDE, A POTENTIAL BACTERICIDAL AGENTJCI Insight, 1973
- Orienting reflex and spectral characteristics of cortical biopotentials in rabbitsNeuroscience and Behavioral Physiology, 1972