Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Zsombor Lacza, James A. Snipes, Jie Zhang, Eszter M. Horváth, Jorge P. Figueroa, Csaba Szabó, David W. Busija

    Research output: Contribution to journalArticle

    115 Scopus citations

    Abstract

    Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.

    Original languageEnglish (US)
    Pages (from-to)1217-1228
    Number of pages12
    JournalFree Radical Biology and Medicine
    Volume35
    Issue number10
    DOIs
    StatePublished - Nov 15 2003

    Keywords

    • Diaminofluorescein
    • Free radicals
    • Mitochondria
    • Nitric oxide synthase
    • mtNOS

    ASJC Scopus subject areas

    • Biochemistry
    • Physiology (medical)

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  • Cite this

    Lacza, Z., Snipes, J. A., Zhang, J., Horváth, E. M., Figueroa, J. P., Szabó, C., & Busija, D. W. (2003). Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. Free Radical Biology and Medicine, 35(10), 1217-1228. https://doi.org/10.1016/S0891-5849(03)00510-0