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Cited by 148Nitric Oxide and Endothelin EffectsHemoglobin structure and functionMetal-Catalyzed Oxygenation Mechanisms

Characterization of bovine endothelial nitric oxide synthase as a homodimer with down-regulated uncoupled NADPH oxidase activity: tetrahydrobiopterin binding kinetics and role of haem in dimerization

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LIST, Barbara M., KLÖSCH, Burkhard, VÖLKER, Christof, GORREN, Antonius C. F., SESSA, William C., WERNER, Ernst R., KUKOVETZ, Walter R., SCHMIDT, Kurt +1 more·1997·Biochemical Journal
DOI 10.1042/bj3230159PMC PMC1218289
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Abstract

The fatty-acylation-deficient bovine endothelial NO synthase (eNOS) mutant (Gly-2 to Ala-2, G2AeNOS) was purified from a baculovirus overexpression system. The purified protein was soluble and highly active (0.2-0.7 μmol of l-citrulline· mg-1·min-1), contained 0.77±0.01 equivalent of haem per subunit, showed a Soret maximum at 396 nm, and exhibited only minor uncoupling of NADPH oxidation in the absence of l-arginine or tetrahydrobiopterin. Radioligand binding studies revealed KD values of 147±24.1 nM and 52±9.2 nM for specific binding of tetrahydrobiopterin in the absence and presence of 0.1 mM l-arginine respectively. The positive co-operative effect of l-arginine was due to a pronounced decrease in the rate of tetrahydrobiopterin dissociation (from 1.6±0.5 to 0.3±0.1 min-1). Low-temperature SDS gel electrophoresis showed that approx. 80% of the protein migrated as haem-containing dimer after preincubation with l-arginine and tetrahydrobiopterin. Gel-filtration chromatography yielded one peak with a Stokes radius of 6.8±0.04 nm, corresponding to a hydrodynamic volume of 1.32×10-24 m3, whereas haem-deficient preparations (approx. 0.3 equivalent per subunit) contained an additional protein species with a hydrodynamic radius of 5.1±0.2 nm and a corresponding volume of 0.55×10-24 m3, suggesting that haem availability regulates eNOS dimerization.

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  • More recent observations indicate that the ADMA/l-arginine ratios in plasma may be a better marker than either metabolite assessed independently.

    “More recent observations indicate that the ADMA/l-arginine ratios in plasma may be a better marker than either metabolite assessed independently (75, 589, 606, 790, 966).”

    From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [589]

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  1. Abu-Soud, H. M. and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10772
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Authors
LIST, B. M., KLÖSCH, B., VÖLKER, C., GORREN, A. C. F., SESSA, W. C., WERNER, E. R., KUKOVETZ, W. R., SCHMIDT, K., & MAYER, B.
Affiliations
  • University of Graz (AT)
  • Yale University (US)
  • Universität Innsbruck (AT)
Corresponding author
  • SCHMIDT, Kurt
  • MAYER, Bernd
ORCID
Venue
Biochemical Journal
Publisher
Portland Press Ltd.
Published
1997-04-01
Volume
323
Issue
1
Pages
159-165
ISSN
  • 0264-6021 (ISSN-L)
  • 1470-8728
DOI
10.1042/bj3230159
PMC
PMC1218289
OpenAlex
W1770314811
Cited by
147
References
61
Citations by year
2025: 1 · 2024: 3 · 2022: 2 · 2021: 4 · 2020: 3 · 2019: 4 · 2018: 4 · 2017: 4 · 2016: 4 · 2015: 7 · 2014: 6 · 2013: 6 · 2012: 3
FWCI
7.73
Type
journal article
Language
en
Open access
Locations
  • Biochemical Journal (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • American Heart Association
  • National Institutes of Health
  • National Heart, Lung, and Blood Institute (HL 57948)
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
  • Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
Keywords
endothelial nitric oxide synthase, homodimerization, tetrahydrobiopterin, heme, L-arginine, protein dimerization, hydrodynamic radius
MeSH terms
Animals, Antioxidants, Biopterins, Cattle, Endothelium, Vascular, Heme, Kinetics, Molecular Weight, Protein Conformation, Spectrophotometry, Atomic, Spodoptera, Nitric Oxide Synthase, NADPH Oxidases, Dimerization

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