Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.
Open accessAbstract
A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.
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- Authors
- Stuehr, D. J., Cho, H. J., Kwon, N. S., Weise, M. F., & Nathan, C. F.
- Affiliations
- Cornell University (US)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 1991-09
- Volume
- 88
- Issue
- 17
- Pages
- 7773-7777
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC52385
- OpenAlex
- W1965077361
- Cited by
- 816
- References
- 33
- Citations by year
- 2026: 2 · 2025: 7 · 2024: 3 · 2023: 9 · 2022: 10 · 2021: 9 · 2020: 8 · 2019: 5 · 2018: 9 · 2017: 4 · 2016: 9 · 2015: 13 · 2014: 14 · 2013: 16 · 2012: 11
- FWCI
- 38.79
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (2r01ca043610-06a1, CA53914)
- Genentech
- National Cancer Institute (CA-53914, CA-43610)
- Topics
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Life Sciences › Immunology and Microbiology › Immunology › Neutrophil, Myeloperoxidase and Oxidative Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
- Keywords
- inducible nitric oxide synthase, macrophages, FAD, flavin mononucleotide, flavoprotein, enzyme purification, interferon-gamma, lipopolysaccharide, tetrahydrobiopterin, L-arginine, NADPH, enzyme dimerization, calmodulin
- MeSH terms
- Amino Acid Oxidoreductases, Animals, Calcium, Calmodulin, Cell Line, Chromatography, Affinity, Chromatography, Gel, Chromatography, High Pressure Liquid, Chromatography, Ion Exchange, Enzyme Induction, Escherichia coli, Flavin-Adenine Dinucleotide, Flavin Mononucleotide, Interferon-gamma, Kinetics, Lipopolysaccharides, Macrophages, Molecular Weight, Cytokines, Nitric Oxide Synthase, Mice
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