Interference with the citrulline‐based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts

There are many reports of an arginine‐dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline‐based assay that meas...

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Bibliographic Details
Published in:The FEBS journal 2007-08, Vol.274 (16), p.4238-4245
Main Authors: Tischner, Rudolf, Galli, Mary, Heimer, Yair M., Bielefeld, Sarah, Okamoto, Mamoru, Mack, Alyson, Crawford, Nigel M.
Format: Article
Language:English
Subjects:
Arabidopsis
Arabidopsis - enzymology
Arabidopsis - metabolism
Arabidopsis Proteins - metabolism
Arginine - metabolism
argininosuccinate lyase
Argininosuccinate Lyase - metabolism
Argininosuccinic Acid - metabolism
Biological Assay - methods
Botany
Catalysis - drug effects
Chromatography, Ion Exchange
Chromatography, Thin Layer
citrulline
Citrulline - metabolism
Enzymes
Flowers & plants
Fumarates - metabolism
Fumarates - pharmacology
Genes
Kinetics
Nitric oxide
Nitric Oxide Synthase - metabolism
Plant Extracts - metabolism
Proteins
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Summary:There are many reports of an arginine‐dependent nitric oxide synthase activity in plants; however, the gene(s) or protein(s) responsible for this activity have yet to be convincingly identified. To measure nitric oxide synthase activity, many studies have relied on a citrulline‐based assay that measures the formation of l‐citrulline from l‐arginine using ion exchange chromatography. In this article, we report that when such assays are used with protein extracts from Arabidopsis, an arginine‐dependent activity was observed, but it produced a product other than citrulline. TLC analysis identified the product as argininosuccinate. The reaction was stimulated by fumarate (> 500 µm), implicating the urea cycle enzyme argininosuccinate lyase (EC 4.3.2.1), which reversibly converts arginine and fumarate to argininosuccinate. These results indicate that caution is needed when using standard citrulline‐based assays to measure nitric oxide synthase activity in plant extracts, and highlight the importance of verifying the identity of the product as citrulline.
ISSN:1742-464X
1742-4658
DOI:10.1111/j.1742-4658.2007.05950.x