Assay of Tissue Activity of Nitric Oxide Synthase

Kurt Schmidt1, Bernd Mayer1

1 Karl‐Franzens‐Universität Graz, Graz, Austria
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 10.2
DOI:  10.1002/0471140856.tx1002s00
Online Posting Date:  May, 2001
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Abstract

Nitric oxide synthase activity can be assayed by a variety of methods. The simplest method to assay activity is the conversion of radiolabeled L‐arginine to L‐citrulline. The assay described in this unit is sensitive, simple to perform, is relatively robust, and can be performed with intact monolayer or suspension cells, or with cell or tissue extracts. The product is easily separated from the reaction by cation‐exchange chromatography.

     
 
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Table of Contents

  • Basic Protocol 1: Determining NOS Activity in Intact Monolayer Cells
  • Alternate Protocol 1: Determining NOS Activity in Intact Suspension Cells
  • Basic Protocol 2: Determining NOS Activity in Cell or Tissue Extracts
  • Support Protocol 1: Preparation and Regeneration of Dowex 50W Chromatography Columns
  • Support Protocol 2: Purify [3H] L‐Arginine by HPLC
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Determining NOS Activity in Intact Monolayer Cells

  Materials
  • Confluent monolayer culture
  • HEPES‐buffered salt solution, pH 7.4 (see recipe), prewarmed to 37°C
  • 10 mM NG‐nitro‐L‐arginine (L‐NNA) or NG‐monomethyl‐L‐arginine (L‐NMA) in water
  • [2,3,4,5‐3H]L‐arginine (HPLC‐purified, see protocol 5) in HEPES‐buffered salt solution (∼10,000 cpm/µl)
  • Stock solution of compound to be tested, prepared as 20× stock solution in HEPES‐buffered salt solution or an appropriate solvent (see step annotation)
  • 0.01 N HCl
  • Scintillation cocktail
  • Sodium acetate buffer with L‐citrulline (see recipe)
  • [3H]L‐citrulline in 0.01 HCl
  • Dowex 50W cation‐exchange columns (for preparation and maintenance of columns, see protocol 4)

Alternate Protocol 1: Determining NOS Activity in Intact Suspension Cells

  Materials
  • Cells or tissue of interest
  • Phosphate‐buffered saline (PBS; appendix 2A), prewarmed to 37°C
  • Triethanolamine buffer with EDTA (see recipe)
  • Assay buffer with [3H]L‐arginine (see recipe)
  • Sodium acetate buffer with EDTA (see recipe)
  • Scintillation cocktail
  • [3H]L‐citrulline in sodium acetate buffer with EDTA (∼10,000 cpm/ml)
  • Ultrasonic generator or mechanical tissue homogenizer (e.g., Polytron, Brinkman)
  • Cheesecloth or 100‐µm‐pore‐size cell strainer
  • Protein assay kit (e.g., Bio‐Rad kit for Bradford protein assay)
  • Dowex 50W cation exchange columns (for preparation and maintenance of columns, see protocol 4)

Basic Protocol 2: Determining NOS Activity in Cell or Tissue Extracts

  Materials
  • Cation‐exchange resin (Dowex 50W, H+ form, 200 to 400 mesh, 8% cross‐linked)
  • Chromatography columns (bed volume 1 to 2 ml, e.g., Poly‐Prep columns from Bio‐Rad)
  • Ethanol
  • 0.5 M and 1 M NaOH
  • 1 M HCl

Support Protocol 1: Preparation and Regeneration of Dowex 50W Chromatography Columns

  Materials
  • HPLC system
  • 50 mM sodium acetate buffer, pH 6.4 ( appendix 2A)
  • [2,3,4,5‐3H]L‐arginine (100 µCi)
  • Scintillation cocktail
  • Methanol
  • HPLC column (Nucleosil 100 10 SA, 4 mm × 250 mm)
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Literature Cited

Literature Cited
   Bredt, D.S. and Snyder, S.H. 1989. Nitric oxide mediates glutamate‐linked enhancement of cGMP levels in the cerebellum. Proc. Natl. Acad. Sci. U.S.A. 86:9030‐9033.
   Castle, J.D. 1995. Purification of Organelles from Mammalian Cells. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P.T. Wingfield, eds.) pp. 4.2.1‐4.2.56. John Wiley & Sons, New York.
   Feelisch, M. and Noack, E.A. 1987. Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur. J. Pharmacol. 139:19‐30.
   Gopalakrishna, R. and Nagarajan, B. 1980. Separation and estimation of arginine‐related metabolites in tissues. Anal. Biochem. 107:318‐323.
   Iyengar, R., Stuehr, D.J., and Marletta, M.A. 1987. Macrophage synthesis of nitrite, nitrate, and N‐nitrosamines: Precursors and role of the respiratory burst. Proc. Natl. Acad. Sci. U.S.A. 84:6369‐6373.
   Mayer, B. and Hemmens, B. 1997. Biosynthesis and action of nitric oxide in mammalian cells. Trends Biochem. Sci. 22:477‐481.
   Murphy, M.E., Piper, H.M., Watanabe, H., and Sies, H. 1991. Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment. J. Biol. Chem. 266:19378‐19383.
   Schmidt, K., Mayer, B., and Kukovetz, W.R. 1989. Effect of calcium on endothelium‐derived relaxing factor formation and cGMP levels in endothelial cells. Eur. J. Pharmacol. 170:157‐166.
   Schmidt, K., Werner, E.R., Mayer, B., Wachter, H., and Kukovetz, W.R. 1992. Tetrahydrobiopterindependent formation of endothelium‐derived relaxing factor (nitric oxide) in aortic endothelial cells. Biochem. J. 281:297‐300.
   Stuehr, D.J. 1997. Structure‐function aspects in the nitric oxide synthases. Annu. Rev. Pharmacol. Toxicol. 37:339‐359.
   Winterbourn, C.C., McGrath, B.M., and Carrell, R.W. 1976. Reactions involving superoxide and normal and unstable haemoglobins. Biochem. J. 155:493‐502.
Key References
   Everse, J. and Grisham, M.B.(eds.) 1995. Methods: A Companion to Methods in Enzymology, Vol. 7: Measurement of Nitric Oxide. Academic Press, San Diego, Calif.
  The following references provide collections of methods used for measuring NO and NOS activity.
   Maines, M.D. (ed.) 1996. Nitric Oxide Synthase: Characterization and Functional Analysis. Academic Press, San Diego, Calif.
   Titheradge, M.A. (ed.) 1998. Methods in Molecular Biology, Vol. 100: Nitric Oxide Protocols. Humana Press, Totowa, N.J.
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