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

类囊体结合 NAD (P) H 脱氢酶的分离和生理作用分析

• 批准号: 06454014
• 负责人: ASADA Kozi
• 金额: $ 3.9万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454014/
• 关键词: Synechocystis; NAD (P) H dehydrogenase; Cyclic electron flow; Cytochrome b 559

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.

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。

项目成果

Shreiber, U.et al.: "Quenching analysis of chlorophyll fluorescence by the saturation pulse method : Particular aspects relating to the study of eukaryotic algae and cyanobacteria" Plant Cell Physiol. 36. 873-882 (1995)

Shreiber，U.等人：“通过饱和脉冲法进行叶绿素荧光的猝灭分析：与真核藻类和蓝细菌研究相关的特定方面”植物细胞生理学。

Sch reiber, U.: "Quenching analysis of Chlorophyll Flurescence by the Saturation pulse method: particalar aspects relating to the study of eukaryotic Algae and Cyanobacteria." Plant and Cell Physiology. 36. 873-882 (1995)

Sch reiber, U.：“通过饱和脉冲法对叶绿素荧光进行猝灭分析：与真核藻类和蓝细菌研究相关的特殊方面。”

Mi, H.et al.: "Thylakoid membrane-bound, NADPH-specific pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the cyanobacterium Synechocystic sp.PCC 6803." Plant Cell Physiol. 36. 661-668 (1995)

Mi, H.等人：“类囊体膜结合的 NADPH 特异性吡啶核苷酸脱氢酶复合物介导蓝藻集胞藻属 sp.PCC 6803 中的循环电子传递。”

Miyake, C.: "Fewedoxin-dependent and Antimycoin A-sensitive reduction of cytochrome b-559 by far-red light in maize thylakoids: paticipation of a menadiol-reducible cytochrome b-559 in cyclic electron flow." Plant and Cell Physiology. 36. 743-748 (1995)

Miyake, C.：“玉米类囊体中远红光对 Fewedoxin 依赖性和 Antimycoin A 敏感的细胞色素 b-559 的还原：甲萘二醇可还原的细胞色素 b-559 在循环电子流中的参与。”

Endo, T. et al.: "Dark induction of non-Photochemical quenching of chlorophyll floures cence by acetate in Chlamydo,onas reinhardtii." Plant and Cell Physiology. 37. in press (1996)

Endo, T. 等人：“莱茵衣藻中乙酸盐对叶绿素荧光的非光化学猝灭的暗诱导”。