Departamento de Bioquímica, Facultad de Veterinaria, Universidad de Córdoba, Córdoba 14005, Spain.
Plant Physiol. 1985 Jul;78(3):614-8. doi: 10.1104/pp.78.3.614.
Homogeneous nitrate reductase (EC 1.6.6.2) from Monoraphidium braunii was obtained by means of affinity chromatography in blue-Sepharose and gel filtration. After electrophoresis in polyacrylamide, gel slices containing pure nitrate reductase were disrupted and injected into previously unimmunized rabbits. The antiserum produced after several weeks was found to inhibit the different activities of nitrate reductase to a similar degree. Monospecificity of the antiserum was demonstrated by Ouchterlony double diffusion and crossed immunoelectrophoresis. The antibodies were purified by immunoabsorption to Sepharose-bound nitrate reductase.The intracellular location of nitrate reductase in green algae was examined by applying an immunocytochemical method to M. braunii cells. Ultrathin frozen sections were first treated with immunopurified anti-nitrate reductase monospecific antibodies, followed by incubation with colloidal gold-labeled goat antirabbit immunoglobulin G as a marker. The enzyme was specifically located in the pyrenoid region of the chloroplast.
从单鞭金藻中通过亲和层析在蓝琼脂糖和凝胶过滤获得均一的硝酸还原酶（EC 1.6.6.2）。在聚丙烯酰胺电泳后，含有纯硝酸还原酶的凝胶片被破坏并注入以前未免疫的兔子体内。数周后产生的抗血清被发现对不同的硝酸还原酶活性具有相似程度的抑制作用。抗血清的特异性通过 Ouchterlony 双扩散和交叉免疫电泳证明。抗体通过免疫吸附到 Sepharose 结合的硝酸还原酶上进行纯化。通过将免疫细胞化学方法应用于单鞭金藻细胞来检查绿藻中的硝酸还原酶的细胞内位置。首先用免疫纯化的抗硝酸还原酶单特异性抗体处理超薄冷冻切片，然后用胶体金标记的兔抗山羊免疫球蛋白 G 孵育作为标记。该酶特异性位于叶绿体的淀粉核区域。
