Lopes Patricia Fátima, de Cabral Oliveira Mariana, Colepicolo Pio
Departamento de Bioquímica, Universidade de São Paulo, Instituto de Química, São Paulo, CP 20780, CEP 05599-970, Brazil.
Biochem Biophys Res Commun. 2002 Jul 5;295(1):50-4. doi: 10.1016/s0006-291x(02)00621-6.
A daily rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to changes in amounts of the protein. The enzyme was purified in four steps: ion exchange Q-Sepharose separation, ammonium sulfate precipitation, gel filtration on Sephacryl S-300, and affinity chromatography on Affigel-blue resin. This purification procedure yielded an active purified NR of about 500-fold with a recovery of 85%. The SDS-PAGE silver staining of purified NR revealed a 110 kDa single band. Non-denaturated protein showed a molecular mass of 440 kDa on gel filtration comparing with SDS-PAGE, the enzyme is apparently composed of four identical subunits. In extracts of algae grown under either constant dim light or a light-dark cycle, the activity of NR exhibited a daily rhythm, peaking at midday phase as does photosynthesis. Staining with monoclonal antibodies, raised against NR from Porphyra yezoensis, showed that the amount of protein changes by a factor of about 12, with a maximum occurring in the midday phase.
从海洋红藻细基江蓠中分离出的硝酸还原酶（NR：EC 1.6.6.1）的活性呈现出日节律，这一现象被证明是由该蛋白质含量的变化所致。该酶通过四个步骤进行纯化：离子交换Q-琼脂糖凝胶分离、硫酸铵沉淀、Sephacryl S-300凝胶过滤以及Affigel-蓝树脂亲和层析。此纯化过程得到了活性纯化的NR，纯化倍数约为500倍，回收率为85%。纯化后的NR经SDS-PAGE银染显示为一条110 kDa的单带。与SDS-PAGE相比，非变性蛋白质在凝胶过滤中显示分子量为440 kDa，该酶显然由四个相同的亚基组成。在持续弱光或明暗周期条件下培养的藻类提取物中，NR的活性呈现出日节律，与光合作用一样在中午阶段达到峰值。用针对条斑紫菜NR产生的单克隆抗体进行染色显示，蛋白质含量变化约12倍，最大值出现在中午阶段。
