Heavy metal induced peptide, Phytochelatin-its biological role and synthetic mechanisms

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1. Purification of Phytochelatin(PC) Z. mays was cultivated in Arnon-lioagland solution containing 3 pM Cd. The root was homogenized with extraction buffer and Cd- containing substance was extracted and purified via gel- filtration and ion exchange chromatographyes. Amino acid analysis of this substance showed Glu:Cys:Gly was 5.9:4.6:1.0, N-terminal and C-terminal amino acids of the substance were indicated to be Glu and Gly, respectively. These data showed Cd-binding substance from Z. mays was composed of PC.2. Determination of PC Analytical method for PC was developed as follows. PCs were separated by HPLC, added by 5,5'-dithiobis(2-nitrobenzoic acid) and detected by the absorption at 412nm. The purified Cd-binding substance was analyzed by this method and shown to consist of seven PCs with different gamma-glutamylcysteine unit. The similar chromatograms were also obtained from the extracts of toots and leaves of Z.mays which were cultivated in Cd-containing medium.3. Detoxification of heavy metals by PC Z.mays was cultivated with Cd and buthioninsulfoximine(BSX). While the growth of the plant was normally in the medium containing Cd or BSX, it was inhibited in the medium containing Cd and BSX. PC was detected with a significant amount in the root cultivated with Cd containing medium, but not detected with the medium containing Cd and BSX. These results indicated that PC was playing an important role on detoxification of heavy metals and that gamma-glutamylcysteine and/or glutatbione could be precursor for PC synthesis.