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

1.NDH活性测定方法：我们利用集胞藻PCC6803分离的类囊体膜建立了NDH活性测定的新方法。NDH的活性可以通过a)叶绿素荧光产量，b)P700的氧化还原变化，c)甲基紫精依赖的Mehler反应，d)在340 nm处的吸光度变化来监测。一种高相对分子质量的酶对NADPH具有特异性，另一种低相对分子质量的酶可同时氧化NADH和NADPH，后者的相对分子质量为30 kDa，对NADPH和NADH的Km值分别为12和47。N端的氨基酸序列与鼠伤寒沙门氏菌硝基还原酶的同源性为46%，而与FNR、DT黄递酶或复合体I亚基的同源性不同。NEM对酶有抑制作用，而鱼藤酮、阿米塔尔、Sham和Diumarol对酶无抑制作用。这些特性，以及与苯醌类似物的高亲和力，表明该酶是一种新的NAD(P)H-苯醌氧化缩合酶。3.高等植物中的循环电子流；我们发现，在光化光关闭后，循环流的活性可以通过叶绿素荧光产量的瞬时增加来监测。玉米体内的循环电子流是由对抗霉素A敏感的细胞色素b_(559)&gt；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.