Isolation and Analysis of Physiological roles of thylakoid-bound NAD (P) H dehydrogenase

1.NDH的测定方法；我们开发了一种新的测量NDH活性的方法，使用从聚囊藻PCC 6803分离的类囊体膜。NDH的活性可以通过a)叶绿素荧光的产率，b) P700的氧化还原变化，c)甲基紫原依赖的Mehler反应，d) 340 nm吸光度变化来监测。NDH的分离；通过活性染色发现两种类型的NDH。一种高分子量的酶对NADPH具有特异性，另一种低分子量的酶对NADH和NADPH都有氧化作用。后一种酶对NADPH和NADH的Km值分别为12和47muM，为30 kDa MW。该酶的n端氨基酸序列与鼠伤寒沙门菌的硝基还原酶有46%的同源性，而与FNR、DT diaphorase和复合体i亚基无同源性，NEM对该酶有抑制作用，鱼tenone、amytal、SHAM和dicumarol对其无抑制作用。这些性质以及与醌类同物的高亲和性表明该酶是一种新型的NAD (P) h -醌氧化还原酶。高等植物的循环电子流；我们发现，在光化光关闭后，叶绿素荧光产量的短暂增加可以监测循环流的活性。还发现玉米循环电子流是由抗霉素A敏感的细胞色素b_<559>介导的。

1.Measuring methods of NDH ; We have developed novel measuring methods of NDH activity using thylakoid membranes isolated from Synechocystis PCC 6803. Activity of NDH can be monitored by a) yield of chlorophyll fluorescence, b) redox change of P700, c) methylviologen-dependent Mehler reaction, d) absorbance change at 340 nm.2.Isolation of NDH ; Two types of NDH were found by activity staining of clude extracts. A high-MW enzyme was specific to NADPH,and the other low-MW enzyme could oxidize both NADH and NADPH.The latter enzyme had 30 kDa MW and showed Km values of 12 and 47muM for NADPH and NADH,respectively. Amino acid sequence of N-terminal showed 46% homology with that of nitroreductase in Salmonella typhimurium, but no homology with that of FNR,DT diaphorase or subunits of complex I.The enzyme was inhibited by NEM,but not by rotenone, amytal, SHAM or dicumarol. These propeeties, along with high affinities with quinone alalogues, indicate that the enzyme was a novel NAD (P) H-quinone oxidoeductase.3.Cyclic electron flow in higher plants ; We found that activity of cyclic flow could be monitored by the transient increase in chlorophyll fluorescence yield after actinic light was turned off. It was also found that cyclic electron flow in maize was mediated by cytochrome b_<559> which was sensitive to antimycin A.