| 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 |
3.212
|
| Times Cited |
12
|
|
WOS Category
|
CHEMISTRY, INORGANIC & NUCLEAR
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| Arabidopsis / Cultured plant cells, genes |
pda00376 |
