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

1. NDH的测定方法：我们已经开发了新的NDH活性的测定方法使用类囊体膜分离从集胞藻PCC 6803。通过叶绿素荧光产率、P700的氧化还原变化、甲基紫精依赖的Mehler反应、340 nm处吸光度的变化，可以监测NDH的活性。2. NDH的分离。一种高分子量的酶对NADPH具有特异性，另一种低分子量的酶可以氧化NADPH和NADPH，后者的分子量为30 kDa，对NADPH和NADH的Km值分别为12和47 μ M。N-端氨基酸序列与鼠伤寒沙门氏菌硝基还原酶的氨基酸序列同源性为46%，与FNR、DT心肌黄酶及复合物I亚基的氨基酸序列均无同源性。该酶能被NEM抑制，但不被鱼藤酮、amytal、SHAM和双香豆素抑制。这些性质，沿着与醌类似物的高亲和力，表明该酶是一种新的NAD（P）H-醌氧化还原酶。3.高等植物中的循环电子流我们发现，在光化光关闭后，叶绿素荧光产量的瞬时增加可以监测循环电子流的活性。同时还发现玉米中的循环电子流是由对抗霉素A敏感的细胞色素B_介导<559>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.