RRC ID 6148
Author Ogawa S, Yoshidomi T, Shirabe T, Yoshimura E.
Title HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators.
Journal J Inorg Biochem
Abstract Phytochelatins (PCs) are nonprotein peptides with the general structure (gamma-Glu-Cys)(n)-Gly (PC(n)), where n is greater than or equal to 2. They are synthesized through a reaction catalyzed by phytochelatin synthase (PCS) in the presence of metal cations and using the tripeptide glutathione (gamma-Glu-Cys-Gly) and/or previously synthesized PC(n) as the substrate. Here, a highly sensitive assay for PCS activity was devised, in which the dequenching of Cu(I)-bathocuproinedisulfonate complexes was used in the detection system of a reversed-phase high-performance liquid chromatograph. Using recombinant PCS from the higher plant Arabidopsis thaliana (rAtPCS1), this assay system was capable of determining PCS activity based on an amount of the enzyme preparation that was 100-fold less than that required for the 5,5'-dithiobis(2-nitrobenzoic acid) assay method. Although adsorption of the enzyme onto the reaction vessel hindered accurate activity determination, the inclusion of bovine serum albumin successfully resolved this issue. This method is a powerful tool for investigating PCS enzyme mechanisms with respect to the roles of metal ions.
Volume 104(4)
Pages 442-5
Published 2010-4-1
DOI 10.1016/j.jinorgbio.2009.12.013
PII S0162-0134(09)00318-3
PMID 20074807
MeSH Aminoacyltransferases / chemistry Aminoacyltransferases / genetics Aminoacyltransferases / metabolism* Animals Arabidopsis Proteins / chemistry Arabidopsis Proteins / genetics Arabidopsis Proteins / metabolism Cattle Chelating Agents / chemistry* Chromatography, High Pressure Liquid / methods* Ions / chemistry* Metals / chemistry* Phytochelatins / biosynthesis Phytochelatins / chemistry Recombinant Proteins / chemistry Recombinant Proteins / genetics Recombinant Proteins / metabolism
IF 3.224
Times Cited 12
Arabidopsis / Cultured plant cells, genes pda00376