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

Authors

  • Rudolf Tischner,

    1.  Albrecht von Haller Institut fur Pflanzenwissenschaften, University of Gottingen, Germany
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    • These authors contributed equally to this work

  • Mary Galli,

    1.  Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA
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    • These authors contributed equally to this work

  • Yair M. Heimer,

    1.  Department of Dryland Biotechnologies, J. Blaustein Institute for Desert Research, Ben-Gurion University, Sede-Boker, Israel
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    • These authors contributed equally to this work

  • Sarah Bielefeld,

    1.  Albrecht von Haller Institut fur Pflanzenwissenschaften, University of Gottingen, Germany
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  • Mamoru Okamoto,

    1.  Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA
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  • Alyson Mack,

    1.  Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA
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  • Nigel M. Crawford

    1.  Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA, USA
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N. M. Crawford, Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0116, USA
Fax/Tel: +1 858 534 1637
E-mail: ncrawford@ucsd.edu

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.

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