Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

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

Research output: Contribution to journalArticle

111 Citations (Scopus)

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
Externally publishedYes

Fingerprint

Mitochondria
Nitric Oxide Synthase
Arginine
Protein Isoforms
Animals
Liver Mitochondrion
Enzymes
Calmodulin
Protein Sorting Signals
Fluorescent Dyes
Immunoprecipitation
Flow cytometry
Confocal Microscopy
Confocal microscopy
Adenosine Diphosphate
Anti-Idiotypic Antibodies
Flow Cytometry
Proteins
Liver
Western Blotting

Keywords

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

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

Cite this

Lacza, Z., Snipes, J. A., Zhang, J., Horváth, E. M., Figueroa, J. P., Szabo, 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

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. / Lacza, Zsombor; Snipes, James A.; Zhang, Jie; Horváth, Eszter M.; Figueroa, Jorge P.; Szabo, Csaba; Busija, David W.

In: Free Radical Biology and Medicine, Vol. 35, No. 10, 15.11.2003, p. 1217-1228.

Research output: Contribution to journalArticle

Lacza, Z, Snipes, JA, Zhang, J, Horváth, EM, Figueroa, JP, Szabo, C & Busija, DW 2003, 'Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS', Free Radical Biology and Medicine, vol. 35, no. 10, pp. 1217-1228. https://doi.org/10.1016/S0891-5849(03)00510-0
Lacza, Zsombor ; Snipes, James A. ; Zhang, Jie ; Horváth, Eszter M. ; Figueroa, Jorge P. ; Szabo, Csaba ; Busija, David W. / Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS. In: Free Radical Biology and Medicine. 2003 ; Vol. 35, No. 10. pp. 1217-1228.
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