HPLC method for the determination of phytochelatin synthase activity specific for soft metal ion chelators.
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.
Ogawa
Shinya
S
Department of Applied Biological Chemistry, School of Agriculture and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan.
Yoshidomi
Takahiro
T
Shirabe
Tomoo
T
Yoshimura
Etsuro
E
D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007477:Ions / Q000737:chemistry*; D008670:Metals / Q000737:chemistry*; D054811:Phytochelatins / Q000096:biosynthesis / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D002614:Chelating Agents; D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D054811:Phytochelatins; D019881:Aminoacyltransferases; C093784:glutathione gamma-glutamylcysteinyltransferase