ELECTRON TRANSPORT PATHWAY MEDIATED BY CHLOROPLASTIC NAD(P)H DEHYDROGENASE

NDH的纯化首先，我试图从菠菜叶中分离叶绿体NDH。由于粗提物中NDH的含量远低于预期，因此很难纯化出足够量的NDH用于酶学研究。接下来，我用一种新开发的叶绿素荧光测定方法筛选了200多种植物的高NDH活性。结果发现，一些C4植物具有很高的NDH活性。因此，我从一些C4植物中纯化了NDH，这些植物可以在市场上买到。从叶片中分离出完整的叶绿体，用去污剂溶解膜相关蛋白复合物。生理分析用NDH K-J和C-K-J亚基失活的叶绿体转化子研究了NDH对强光的反应。基质和系统间链的氧化还原状态分别用P700氧化还原和叶绿素荧光进行监测。提出了NDH在防止强光照射后诱导的过度还原中的关键作用。

Purification of NDHFirst, I tried to isolate chloroplastic NDH from spinach leaves. The content of NDH in the crude extract was very much lower than expected thus I concluded that it would be very difficult to purify enough amount of NDH for enzymological study. Next, I screened more than 200 plant species for high NDH activity using a newly developed assay method with chlorophyll fluorescence. As a results I found that some C4 plants have very high NDH activity. Therefore I purified NDH from a few C4 plant species which could be commercially available. Intact chloroplasts were isolated from leaves, and membrane associated protein complexes were solubilized by detergents. Then NDH was purified through anion exchange and gel filtration chromatographies.Physiological analysisResponses to very high light was investigated using newly generated chloroplast transformants in which NDH K-J or C-K-J subunits were inactivated. Redox states in stroma and the intersystem chain were monitored with P700 redox and chlorophyll fluorescence, respectively. A crucial role of NDH in preventing over-reduction induced after exposure to high light was proposed.