Open AccessNitrate Reductase from Monoraphidium braunii
Author(s) -
Arnaldo LopezRuiz,
J. Roldán,
JeanPierre Verbelen,
Jesús Dı́ez
Publication year - 1985
Publication title -
plant physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.554
H-Index - 312
eISSN - 1532-2548
pISSN - 0032-0889
DOI - 10.1104/pp.78.3.614
Subject(s) - nitrate reductase , sepharose , biochemistry , antiserum , ouchterlony double immunodiffusion , nitrate , reductase , biology , enzyme , immunoelectrophoresis , affinity chromatography , size exclusion chromatography , polyacrylamide gel electrophoresis , chromatography , chemistry , microbiology and biotechnology , antibody , ecology , immunology
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.