Nitrate Reductase from Monoraphidium braunii: Immunocytochemical Localization and Immunological Characterization

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.