title: Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts.
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 microM), 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.
authors.affiliation: Albrecht von Haller Institut fur Pflanzenwissenschaften, University of Gottingen, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tischner
authors.forename: Mary
authors.initials: M
authors.lastname: Galli
authors.forename: Yair M
authors.initials: YM
authors.lastname: Heimer
authors.forename: Sarah
authors.initials: S
authors.lastname: Bielefeld
authors.forename: Mamoru
authors.initials: M
authors.lastname: Okamoto
authors.forename: Alyson
authors.initials: A
authors.lastname: Mack
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Crawford
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism; D001123:Argininosuccinate Lyase / Q000378:metabolism*; D001125:Argininosuccinic Acid / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D002384:Catalysis / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D002956:Citrulline / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010936:Plant Extracts / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D005650:Fumarates; D010936:Plant Extracts; D001125:Argininosuccinic Acid; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase; D001123:Argininosuccinate Lyase