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

1.植物络合蛋白(PC)的纯化。在含有3 PM Cd的Arnon-Lioagland溶液中培养玉米。用提取缓冲液匀浆，凝胶过滤和离子交换层析提取纯化含镉物质。该物质的氨基酸组成为：Glu：Cys：Gly=5.9：4.6：1.0，N-端为Glu，C-端为Gly。这些数据表明Z.Mays的Cd结合物质是由PC.2组成的。PC的测定发展了PC的分析方法如下。用高效液相色谱分离，加入5，5‘-二硫代二(2-硝基)苯甲酸，在412 nm处进行检测。用该方法对纯化的Cd结合物质进行了分析，结果表明，纯化的Cd结合物质由7个不同γ-谷氨酰半胱氨酸单元的PC组成。在含Cd的培养液中，麦草的根和叶的提取物也得到了类似的色谱图。用Cd和丁硫基胰岛素亚胺(BSX)培养PCZ对重金属的解毒作用。植株在含Cd或BSX的基质中正常生长时，在含Cd和BSX的基质中生长受到抑制。用Cd+BSX培养的根中检测到大量的PC，而用Cd+BSX培养的根中没有检测到PC。这些结果表明PC在重金属的解毒中起着重要的作用，γ-谷氨酰半胱氨酸和/或谷氨二酮可能是PC合成的前体。

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.