Moureaux T, Leydecker M T, Meyer C
Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Versailles, France.
Eur J Biochem. 1989 Feb 15;179(3):617-20. doi: 10.1111/j.1432-1033.1989.tb14591.x.
Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).
使用5'-AMP-琼脂糖层析法或两步免疫亲和层析法从垂花烟草叶片中纯化硝酸还原酶，两步免疫亲和层析法涉及针对玉米硝酸还原酶和垂花烟草核酮糖-1,5-二磷酸羧化酶的单克隆抗体。通过第一种方法获得的硝酸还原酶纯化了1000倍，比活性为9单位/毫克蛋白质。第二种方法产生了一种纯酶，纯化了21000倍，比活性为80单位/毫克蛋白质。硝酸还原酶的SDS/PAGE总是产生两条带，分别为107 kDa和99.5 kDa。107 kDa的条带是垂花烟草的硝酸还原酶亚基；如通常报道的那样，较小的99.5 kDa条带被认为是较大蛋白质的蛋白水解产物。107 kDa的分子量与最近从烟草（Nicotiana tabacum cv Xanthi）克隆的两个硝酸还原酶基因的编码序列计算的值接近。
