Cited by 816Nitric Oxide and Endothelin EffectsNeutrophil, Myeloperoxidase and Oxidative MechanismsRedox biology and oxidative stress

Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein.

Open access
Stuehr, D J, Cho, H J, Kwon, N S, Weise, M F, Nathan, C F·1991·Proceedings of the National Academy of Sciences

Abstract

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.

Integrity signals

Registry

Crossref · 10.1073/pnas.88.17.7773

Cites

33 of 33 references verified0 citation errors · 0 not checkable

Statistics

Recomputing p-values needs the paper’s full text.

Claims the paper makes

The text we found for this paper looks like another document, so its claims and self-check weren’t run. The registry and citation checks still apply.

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The text we found for this paper looks like another document, so the self-check wasn’t run.

Cites

Flagged first, then by citations

Cited by

Sorted by citations

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
Published
1991-09
Volume
88
Issue
17
Pages
7773-7777
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
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
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

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review