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

Rudolf Tischner, Mary Galli, Yair M. Heimer, Sarah Bielefeld, Mamoru Okamoto, Alyson Mack, Nigel M. Crawford

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    42 Scopus citations

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)4238-4245
    Number of pages8
    JournalFEBS Journal
    Volume274
    Issue number16
    DOIs
    StatePublished - 1 Aug 2007

    Keywords

    • Arabidopsis
    • Argininosuccinate lyase
    • Citrulline
    • Nitric oxide

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