Nitric Oxide Synthase Assays

Jie Zhang1

1 Guilford Pharmaceuticals, Baltimore, Maryland
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 2.4
DOI:  10.1002/0471141755.ph0204s00
Online Posting Date:  May, 2001
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Abstract

Nitric oxide (NO) is a widespread messenger molecule that regulates cardiovascular tone, immune responses, and neuronal function. This unit provides a simple protocol for measuring NO production based on the stoichiometric formation of the byproduct L‐citrulline by nitric oxide synthase (NOS) when NO is produced. Support protocols describe the preparation and purification of NOS from tissues or cultured cells, and for the purification of the radiolabeled L‐arginine substrate by thin‐layer chromatography (TLC).

     
 
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Table of Contents

  • Basic Protocol 1: Arginine‐to‐Citrulline Conversion Assay for Measuring NOS Activity
  • Support Protocol 1: Preparation of NOS‐Containing Tissue Extracts
  • Support Protocol 2: One‐Step Partial Purification of NOS
  • Support Protocol 3: Preparation of NOS from Cultured Cells
  • Support Protocol 4: Purification of [3H]Arginine by Thin‐Layer Chromatography
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Arginine‐to‐Citrulline Conversion Assay for Measuring NOS Activity

  Materials
  • ∼50% (v/v) Dowex AG50WX‐8 in water (resin slurry; use Na+ form)
  • Stop solution (see recipe)
  • 10× reaction buffer (see recipe)
  • 10 mM NADPH (8.33 mg/ml; prepare fresh; e.g., Sigma)
  • 50 µCi/ml L‐[U‐14C]arginine monohydrochloride ([14C]Arg; 300 mCi/mmol; Amersham) or TLC‐purified [3H]arginine ([3H]Arg; see protocol 5)
  • NOS‐containing extracts from tissue or cell culture (see protocol 2Support Protocol 1, protocol 32, or protocol 43)
  • 10 mM aqueous Nω‐nitro‐L‐arginine methyl ester HCl (NAME; store aliquots at −20°C)
  • Fritted disposable columns (e.g., Bio‐Rad)
NOTE: Some vendors (e.g., Sigma) sell the protonated (H+) form of the Dowex resin. This can be converted to the sodium salt (Na+) form by adding NaOH pellets to the resin until the pH is >12, equilibrating for 1 hr, and then bringing the pH back to <8 through sequential batch‐washing with H 2O.

Support Protocol 1: Preparation of NOS‐Containing Tissue Extracts

  Materials
  • NOS‐containing tissue (e.g., rat cerebellum)
  • Homogenization buffer (see recipe), ice cold
  • Extraction buffer (see recipe), ice cold
  • Homogenizer (e.g., Polytron from Brinkmann)

Support Protocol 2: One‐Step Partial Purification of NOS

  Materials
  • 2′,5′‐ADP‐agarose (e.g., Sigma)
  • No‐salt and high‐salt washing solution (see recipe), ice‐cold
  • Elution buffer (see recipe), ice‐cold
  • Fritted disposable columns (e.g., Bio‐Rad)
  • Additional reagents and equipment for preparation of tissue extract (see protocol 2)
NOTE: Perform all steps at 4°C.

Support Protocol 3: Preparation of NOS from Cultured Cells

  Materials
  • NOS‐containing cell culture, fresh
  • PBS (see recipe), ice cold
  • Homogenization buffer or extraction buffer (see reciperecipes), ice cold
  • Tissue grinder or sonicator

Support Protocol 4: Purification of [3H]Arginine by Thin‐Layer Chromatography

  Materials
  • 1 µCi/µl [2,3,4,5‐3H]arginine ([3H]Arg)
  • TLC standards (1 mM each Arg, Cit, and ornithine; prepare fresh)
  • Developer (90 ml methanol + 15 ml ammonium hydroxide; prepare fresh)
  • 2 mg/ml ninhydrin solution in 3% acetic acid (prepare fresh and place in a spray bottle)
  • 0.1 M HCl
  • Nonradioactive L‐arginine (Arg)
  • 20 × 20–cm silica‐gel TLC plate
  • TLC tank
  • 0.5 × 10–cm polystyrene disposable chromatography columns (Evergreen Scientific)
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Figures

Videos

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.
   Bredt, D.S. and Snyder, S.H. 1990. Isolation of nitric oxide synthetase, a calmodulin‐requiring enzyme. Proc. Natl. Acad. Sci. U.S.A. 87:682‐685.
   Bredt, D.S., Hwang, P.M., and Snyder, S.H. 1990. Localization of nitric oxide synthase indicating a neuronal role for nitric oxide. Nature 347:768‐770.
   Bredt, D.S., Hwang, P.M., Glatt, C.E., Lowenstein, C., Reed, R.R., and Snyder, S.H. 1991. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P‐450 reductase. Nature 351:714‐718.
   Frey, C., Narayanan, K., McMillan, K., Spack, L., Gross, S., Masters, B.S., and Griffith, O.W. 1994. L‐Thiocitrulline: A stereospecific, heme‐binding inhibitor of nitric oxide synthase. J. Biol. Chem. 269:26083‐26091.
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Key References
   Bredt and Snyder, 1989. See above.
  Original paper demonstrating that citrulline formation parallels NO‐stimulated cGMP accumulation, and thereby establishing the basis for measuring citrulline formation as a NOS assay.
   Bredt and Snyder, 1990. See above.
  This paper showed the high efficiency of using the arginine‐to‐citrulline assay to rapidly quantify NOS activity.
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