Synthetic mechanisms of Cd-induced peptides in fusion yeast

1. 采用ODS柱高效液相色谱分离，DTNB衍生化，412 nm吸收测定的方法，建立了植物螯合肽的高灵敏度测定方法。它可以测定谷胱甘肽和PCs，每个样品约15 sin。在无Cd条件下培养pombe细胞，测定PC2及其相关化合物的浓度。在培养早期，PC2水平升高，同时谷胱甘肽含量降低。说明PC是通过谷胱甘肽合成的。以无Cd培养的s.p ebe细胞为原料，制备了pc合成酶粗提液，并采用硫酸铵分离和疏水层析对其活性进行了纯化。将含合成酶的馏分加到羟基磷灰石柱上。然而，这种活动却消失了。此外，人们发现这种酶非常不稳定，因此一种防止这种酶失活的贮藏方法目前仍在研究中。

1. Development of high sensitive determination of PC Phytochelatins were determined by such a way that each PC was separated by HPLC with ODS column, derivatized with DTNB and determined by absorption at 412 nm. It enables to determine glutathione and PCs about 15 sin per a sample.2. Change in PC and its related compounds in culturing Concentrations of PC and its related compounds were measured when S. pombe cells were cultured in the absence of Cd. In the early culturing period the level of PC2 is increased with a concomitant decrease in glutathione. These indicate that PC is synthesized via glutathione.3. Purification of PC-synthsizing enzymeA crude extract solution was prepared from S. poebe cells which were cultured in the absence of Cd. The activity was purified by ammonium sulfate fractionation and hydrophobic chromatography. The fraction containing the synthetic enzyme was applied to a hydroxy appatite column. The activity was, however, lost. In addition the enzyme was found to be very unstable so that a straging method which prevent the enzyme from inactivating is now still on investigation.