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

首先，我尝试从菠菜叶中分离出叶绿体NDH。粗提物中NDH的含量远远低于预期，因此我认为很难纯化出足够量的NDH进行酶学研究。接下来，我用一种新开发的叶绿素荧光检测方法筛选了200多种高NDH活性的植物。结果我发现一些C4植物具有非常高的NDH活性。因此，我从一些C4植物中纯化了NDH，这些植物可以在商业上使用。从叶片中分离出完整的叶绿体，并用洗涤剂溶解膜相关蛋白复合物。然后通过阴离子交换和凝胶过滤层析纯化NDH。利用失活NDH K-J或C-K-J亚基的新生成的叶绿体转化体，研究了对强光的响应。利用P700氧化还原和叶绿素荧光分别监测基质和系统间链的氧化还原状态。NDH在防止强光照射后的过度还原中起着至关重要的作用。

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.