Kubo Y, Ogura N, Nakagawa H
Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Japan.
J Biol Chem. 1988 Dec 25;263(36):19684-9.
The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.
通过有限蛋白酶解实验研究了菠菜叶片同化型NADH-硝酸还原酶的功能结构。用胰蛋白酶孵育纯化的硝酸还原酶后，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳观察到59 kDa和45 kDa的两种稳定产物。通过Blue Sepharose色谱法纯化了45 kDa的片段。NADH-铁氰化物还原酶和NADH-细胞色素c还原酶活性与这个包含FAD、血红素和NADH结合片段的45 kDa片段相关。用金黄色葡萄球菌V8蛋白酶孵育纯化的硝酸还原酶后，通过高效液相色谱尺寸排阻凝胶过滤观察到两个主要峰。FMNH2-硝酸还原酶和还原型甲基紫精-硝酸还原酶活性与170 kDa的第一个峰相关，该峰由两个非共价结合的（75 - 90 kDa）片段组成。然而，NADH-铁氰化物还原酶活性与由FAD和NADH结合位点组成的第二个峰相关。用金黄色葡萄球菌V8蛋白酶孵育45 kDa的片段产生了分别包含FAD和血红素的28 kDa和14 kDa的两个主要片段。这些结果表明，菠菜硝酸还原酶的钼、血红素和FAD组分包含在由暴露的铰链区共价连接的不同结构域中。钼结构域似乎在维持酶复合物中的亚基相互作用方面很重要。
