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Cited by 12Nitric Oxide and Endothelin EffectsAmino Acid Enzymes and MetabolismMitochondrial Function and Pathology

Arginase activity in mitochondria – An interfering factor in nitric oxide synthase activity assays

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Venkatakrishnan, Priya, Nakayasu, Ernesto S., Almeida, Igor C., Miller, R.T.·2010·Biochemical and Biophysical Research Communications
DOI 10.1016/j.bbrc.2009.10.169PMC PMC2854296
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Abstract

Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [(14)C]-L-arginine to [(14)C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent. Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [(14)C]-L-arginine to [(14)C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

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  • In the NOS activity assay, urea can be coeluted with L-citrulline and provide a false-positive signal for NOS activity.

    “Briefly, in this NOS activity assay, urea can be coeluted with L-citrulline and thereby provide a false-positive signal for NOS activity.”

    From Redox regulation of mitochondrial function (2012) · cites this paper as [432]

  • The NOS activity assay measuring radiolabeled [14C]-L-Arg catabolism to L-citrulline can also detect arginase-mediated urea production if insufficient arginase inhibitors are used.

    “A subsequent study by this group reported that one of the commonly used NOS activity assays that measures radiolabeled [14C]-L-Arg catabolism to L-citrulline, a byproduct of NO production, can also detect arginase-mediated urea production if insufficient levels of arginase inhibitors are used (432).”

    From Redox regulation of mitochondrial function (2012) · cites this paper as [432]

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Authors
Venkatakrishnan, P., Nakayasu, E. S., Almeida, I. C., & Miller, R. T.
Affiliations
The University of Texas at El Paso (US)
Corresponding author
Miller, R.T.
ORCID
Venue
Biochemical and Biophysical Research Communications
Publisher
Elsevier BV
Published
2010-04
Volume
394
Issue
3
Pages
448-452
ISSN
0006-291X (ISSN-L)
DOI
10.1016/j.bbrc.2009.10.169
PMC
PMC2854296
OpenAlex
W2049842976
Cited by
12
References
40
Citations by year
2018: 1 · 2017: 1 · 2016: 2 · 2015: 1 · 2014: 2 · 2013: 2 · 2012: 1
FWCI
0.66
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemical and Biophysical Research Communications (publishedVersion)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Center for Research Resources
  • National Institutes of Health (5G12RR008124)
  • National Institute of Environmental Health Sciences
Topics
  • Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Amino Acid Enzymes and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
Keywords
mitochondrial nitric oxide synthase, arginase, L-arginine, citrulline, arginase inhibitors, rat liver mitochondria
MeSH terms
Animals, Arginase, Chromatography, High Pressure Liquid, Chromatography, Thin Layer, Mitochondria, Liver, Reproducibility of Results, Nitric Oxide Synthase, Rats

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