tailieunhanh - Báo cáo khoa học: 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 syn-thase activity, many studies have relied on a citrulline-based assay that measures the formation ofl-citrulline from l-arginine using ion exchange chromatography. | ễFEBS Journal Interference with the citrulline-based nitric oxide synthase assay by argininosuccinate lyase activity in Arabidopsis extracts Riiflftlf Ticrhnot 1 f Qlli2 Vair l l l-loimot 3 R i ft I ftf ft I fl1 n r 11 Cil amHtH2 nuuon lisciiner iviaiy Galli Tall IVI. Heimer SaiaH Dieleield Mamoru Okamoto Alyson Mack2 and Nigel M. Crawford2 1 Albrecht von Haller Institut fur Pflanzenwissenschaften University of Gottingen Germany 2 Section of Celland DevelopmentalBiology Division of BiologicalSciences University of California at San Diego La Jolla CA USA 3 Department of Dryland Biotechnologies J. Blaustein Institute for Desert Research Ben-Gurion University Sede-Boker Israel Keywords Arabidopsis argininosuccinate lyase citrulline nitric oxide Correspondence N. M. Crawford Section of Celland DevelopmentalBiology Division of BiologicalSciences University of California at San Diego La Jolla CA 92093-0116 USA Fax Tel 1 858 534 1637 E-mail ncrawford@ These authors contributed equally to this work Received 23 February 2007 revised 24 May 2007 accepted 20 June 2007 doi 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 pM implicating the urea cycle enzyme argininosuccinate lyase EC which reversibly converts arginine and fumarate to argi-ninosuccinate. These results indicate that caution is needed when using standard